Dirk Görlich
Max Planck Institute
Steven L. McKnight
UT Southwestern Medical Center
For discoveries that exposed the structures and functions of low-complexity domains within protein sequences, revealing new principles of intracellular transport and cellular organization
The 2025 Albert Lasker Basic Medical Research Award honors two scientists for discoveries that exposed the structures and functions of low-complexity domains (LCDs) within protein sequences. With bold imagination and ingenious experimentation, Dirk Görlich (Max Planck Institute for Multidisciplinary Sciences, Göttingen) and Steven L. McKnight (UT Southwestern Medical Center, Dallas) revealed new principles of intracellular transport and cellular organization. Textbooks spotlight proteins for their structural complexity and chemical sophistication, stressing the unique three-dimensional structures that allow these molecules to perform specialized jobs. Their diverse properties arise from the order of their amino acids, each of which confers idiosyncratic characteristics. In this conventional view, LCDs, which are composed of just a few amino acids rather than the full complement of 20, flop around, with little capacity to do important tasks. Yet approximately 15-20% of eukaryotic proteins contain such tracts, and Görlich and McKnight have shown that these “disordered” LCD domains coalesce within cells to support manifold physiological operations.
Dissolving the nuclear transport paradox
Eukaryotic cells hold numerous membrane-bound compartments that collect the machinery for distinct activities. Among these conventional organelles are nuclei, whose surrounding membrane separates their contents from that of the cytoplasm. Molecules move between the two chambers via a highly selective transport system that relies on nuclear pore complexes. These protein channels penetrate the nuclear membrane and create conduits through which nucleocytoplasmic exchange occurs.
By the turn of the 21st century, key features of import into the nucleus had emerged. Small molecules can get through the pores without help, but large ones can’t. Their passage requires transporter proteins. As a postdoctoral fellow, Görlich identified one of these transporters—the first known factor that helps relocate proteins from the cytoplasm, through the nuclear pore, into the nucleus.
Scientists knew that transporters also interact with proteins that pack the center channel of the pores and that these proteins, nucleoporins, contain regions of low amino-acid complexity that are peppered with a two amino-acid motif, phenylalanine (F)-glycine (G). Furthermore, these LCDs, dubbed “FG repeats,” facilitate translocation of the transporters, with or without loaded cargo. The mechanism, however, was opaque.
These observations raised conundrums. Associating with transporters hastens nuclear entry of cargo even though the combination is larger than the cargo alone. Moreover, binding of transporters to anything—in this case, nucleoporins—should delay travel, not accelerate it.
He conceived a model in which the nucleoporin’s low-complexity domains—hydrophobic by nature, due in part to their many phenylalanines—grab one another to minimize water exposure. Large molecules can slide through the hydrophobic wall only if they overcome the repulsion (Figure, part A).
Görlich proposed that transporters bind to the nucleoporin LCDs and thus compete with inter-repeat associations; consequently, amino acids in the LCDs disengage from one another and instead embrace the transporters. In this way, transporters melt into the mesh, which seals around the migrating entity and remains an obstruction to other macromolecules even when a bulky item is inside the channel. As the transporters shuttle through the nuclear pore, hydrophobic bonds re-fasten behind them. This “selective solvation” explains the facilitated movement as well as the blockade to molecules that can’t partition into the nucleoporin sieve. The permeability and barrier mechanisms are intrinsically linked. Görlich set out to interrogate this visionary scheme.
A hypothesis gels
The following year, Görlich reported that transporters are unusually hydrophobic and that disrupting the hydrophobicity of the nuclear pore makes it open. These developments affirmed the influence of hydrophobicity on sorting, but they did not directly demonstrate the existence of the material within the pore that he had posited.
He produced just the LCD of a well-known nucleoporin—and then, under certain conditions, something surprising and exciting happened: It solidified into a translucent gel, the type of matter that he had predicted. Through genetic engineering, he changed each phenylalanine to serine, a nonhydrophobic amino acid. The resulting protein remained liquid, suggesting that hydrophobicity contributes to gel formation.
The gel mimicked a nuclear pore’s discernment. It excluded test proteins that cannot pass through a pore on their own, yet allowed multiple transporters to penetrate and spread within. A large cargo protein remained almost completely outside unless it united with a transporter.
These results, published in 2006 and 2007, were astounding, but questions remained. In particular, no one had established that transit through intact nuclear pore complexes relies on cohesion among the LCD-containing nucleoporins—a central element of Görlich’s model. He wanted to scrutinize his ideas in the context of a real nuclear pore complex.
He reconstituted nuclear pores from frog egg extracts and, in 2012, showed that the permeability barrier requires hydrophobic interactions among the phenylalanine-rich repeats. These experiments bolstered his hypothesis and ruled out other leading theories of the time.
Order from disorder
A. The central channel of the nuclear pore complex (top-left) is packed with the low-complexity domains (LCDs) of nucleoporins, called FG repeats. These FG repeats create a permeability barrier through which transporter proteins—with or without cargo—move between the cytoplasm and the nucleus (only nuclear influx is shown). Other macromolecules are excluded. Hydrophobic interactions among the repeats (top-right) form the basis of this selective transport through the nuclear pore. Transporter proteins, which are also hydrophobic, displace these inter-repeat associations to melt into—and through—the channel of the nuclear pore complex.
B. In RNA granules, RNA-binding proteins associate through their low-complexity domains (bottom-left). Hydrogen bonds among the proteins (bottom-right) create cross-beta interactions that facilitate transient connections, allowing the granules to assemble and disassemble as needed inside cells.
Illustration: Cassio Lynm / © Amino Creative
Three years later, Görlich reported that the LCD of a nucleoporin that is found in diverse creatures—animals, plants, fungi, and protists—spontaneously assembles into dense particles that grant access to transporters and their cargo. These floating nuclear pores resembled liquid droplets that temporarily form inside cells to serve vital roles.
Görlich crowned his nuclear-pore work with an elegant and powerful 2021 publication. In it, he demonstrated that a simple synthetic protein that contains FG repeats recapitulates selective nuclear import. He had distilled the barrier to its essence, producing the most relevant properties with a 12-amino-acid LCD sequence, repeated 52 times.
Low complexity, high impact
McKnight studied gene regulation, not nuclear pores, but he had noticed Görlich’s papers about the unusual gelling of the nucleoporins. A serendipitous observation led him to kindred self-assembling substances. While seeking molecular partners of a chemical that stimulates embryonic stem cells to turn into cardiac muscle, he and collaborator Masato Kato mixed a derivative of this isoxazole-based compound with mashed-up cells. Hundreds of proteins stuck to it. This result initially seemed disastrous: The chemical apparently bound proteins nonspecifically. But McKnight noticed that many of the captured molecules were RNA-binding proteins that populate RNA granules.
One common feature popped out. Each protein includes a domain of low sequence complexity. As in the nucleoporins, these segments deploy only a few of the 20 amino acids, although different ones. McKnight had grappled with low-complexity domains (LCDs) decades earlier because, in many proteins, they bestow the ability to activate genes. Illuminating the function of these wiggly, disordered spans–as they exist when extracted from cells–had stumped him. Now he had arrived back at LCDs by accident.
To assess whether the LCDs underlie the association with the isoxazole-based compound, he and Kato removed that stretch of amino acids from several RNA-binding proteins or added the clipped off portion to a test molecule that normally ignores the chemical. The LCD was necessary and sufficient for binding. While conducting these experiments, they noticed that FUS, one of the LCD-containing proteins, gels—just like Görlich’s nucleoporins. Further investigation established that the LCD domain rules this property too. McKnight published these findings in 2012.
The impact of McKnight’s work was immediate because it explained in precise terms the basis for membrane-less, RNA-rich organelles that behave like liquid droplets, an observation reported by Clifford Brangwynne, Anthony Hyman (both then at the Max Planck Institute for Molecular Cell Biology and Genetics, Dresden), and Frank Jülicher (Max Planck Institute for the Physics of Complex Systems, Dresden). In an important 2009 paper, they established that Görlich’s principle of phase separation applies to a type of RNA-rich, membrane-less organelle called the P granule—and that this particle rapidly dissolves and condenses. The researchers proposed that other self-assembling units rely on a similar process, but they did not address an underlying molecular mechanism to explain it.
McKnight’s electron microscopy images of LCD-composed gels revealed long, uniform fibers, and X-ray diffraction presented hallmark attributes of the so-called cross-beta associations that characterize amyloid fibers found in pathogenic conditions such as Alzheimer disease. In this construction strategy, constituent protein chains stack by virtue of hydrogen bonds between their backbones. However, unlike classic amyloids, which boast exceptional toughness, FUS conglomerations succumbed to detergent, which dismantled them.
In the meantime, McKnight had noticed that the isoxazole derivative forms a crystal whose surface displays a series of troughs with a distinctive pattern of hydrogen bond donors and acceptors that could perfectly accommodate unfurled protein strands. In the 2012 paper that shared these results, he proposed that the LCDs exploit related configurations within cells to cluster into RNA granules. In his view, the necessary LCD associations are less extensive and more fleeting than those in gels, yet they share the same chemical basis. Reversible coalescence would enable functional centers to organize when appropriate and fall apart after use (Figure, part B).
For this scenario to hold up, McKnight needed to substantiate the biological relevance of the LCD interactions. Toward that end, he probed the LCD of an RNA-binding protein (hnRNPA2) in gels and in nuclei from mammalian cells by chemically adorning exposed portions. In 2015, he reported that it adopts a similar structure in both settings as well as in liquid droplets.
To pin down its atomic details, he teamed up with biophysicist Robert Tycko (NIH). Solid state nuclear magnetic resonance spectroscopy exhibited a cross-beta core within FUS’s LCD.
The case continued to build that LCDs consort with one another through these connections in healthy living cells to perform crucial feats. For example, in 2021, McKnight and Tycko showed that the LCDs within components of the cell’s skeleton—intermediate filaments—assume this arrangement in their normally organized state.
The power of weakness
In 2022, through a novel experimental approach, McKnight directly tested whether hydrogen bonds between protein backbones—a cardinal piece of cross-beta architecture—contribute to LCD assembly. Manipulating such hydrogen bonds is not trivial, as standard genetic engineering methods alter side chains, not the backbone. McKnight blocked hydrogen bonding by chemically capping nitrogen atoms in the backbone and measured whether this procedure interfered with the ability of the LCDs to congregate. He focused on a region of an LCD that is essential for phase separation.
These experiments and others mapped precise spots at which main-chain hydrogen bonds foster phase separation, thus cementing their involvement in the process and adding weight to the idea that intracellular LCD fraternization depends on cross-beta connections. Furthermore, the observation that single hydrogen bonds deliver measurable effects confirmed the principle that self association sits on a threshold that can tilt one way or the other. Capturing these short-lived cross-beta associations is extremely challenging and demands the type of rigorous experimentation that McKnight undertook.
Notably, the amino acid proline cannot participate in hydrogen bonding because its main-chain nitrogen atom does not possess the necessary hydrogen. Perhaps, McKnight reasoned, proline could interrupt extended trains of cross-beta hydrogen bonds and thus protect against toxic aggregation. He wondered whether disease-causing mutations that substitute another amino acid for proline in LCDs might corroborate this notion.
To explore this possibility, he focused on point mutations that replace prolines in the genes encoding three different proteins (NFL, neurofilament light chain; tau; and hnRNPA2) that cause, respectively, three different diseases: the inherited nerve disorder Charcot-Marie-Tooth disease, frontotemporal dementia, and Paget’s disease. Peptides with the troublemaking amino acids aggregated in test-tube studies, and blocking the backbone hydrogen-bonding ability of the offending amino acids restored normal behavior. These results suggest that mutations drive pathogenesis of these illnesses by stabilizing normally reversible cross-beta connections.
The inherently weak associations among LCD-containing proteins confer profound benefits. Many physiological processes must toggle, operating when appropriate and ceasing as conditions change. Transient interactions lend themselves to regulatory mechanisms that nudge them in one direction or the other. Validating such ethereal events poses extraordinary challenges, but despite these obstacles, the LCD picture has prevailed.
McKnight and Görlich have transformed our understanding of a fundamental aspect of biology by showing that a significant fraction of the proteome—those 15-20% of proteins with LCDs—enable cells to create flexible, reversible structures that organize and regulate function beyond traditional membrane-bound organelles.
by Evelyn Strauss
Selected Publications – Dirk Görlich
Görlich D, Prehn S, Laskey RA, and Hartmann E. (1994). Isolation of a protein that is essential for the first step of nuclear protein import. Cell. 79, 767-778.
Frey S, Richter RP, and Görlich D. (2006). FG-rich repeats of nuclear pore proteins form a three-dimensional meshwork with hydrogel-like properties. Science. 314, 815-817.
Frey S and Görlich D. (2007). A saturated FG-repeat hydrogel can reproduce the permeability properties of nuclear pore complexes. Cell. 130, 512-523.
Mohr D, Frey S, Fischer T, Güttler T, and Görlich D. (2009). Characterisation of the passive permeability barrier of nuclear pore complexes. EMBO J. 28, 2541-2553.
Ader C, Frey S, Maas W, Schmidt HB, Görlich D, and Baldus, M. (2010). Amyloid-like interactions within nucleoporin FG hydrogels. Proc. Natl. Acad. Sci. USA. 107, 6281-6285.
Schmidt HB and Görlich D. (2016). Transport selectivity of nuclear pores, phase separation, and membraneless organelles. Trends Biochem Sci. 41, 46-61.
Ng SC, Güttler T, and Görlich D. (2021). Recapitulation of selective nuclear import and export with a perfectly repeated 12mer GLFG peptide. Nat Commun. 12, 4047. doi.org/10.1038/s41467-021-24292-5.
Selected Publications – Steven L. McKnight
Kato M, Han TW, Xie S, Shi K, Du X, Wu LC, Mirzaei H, Goldsmith EJ, Langgood J, Pei J, Grishin NV, Frantz DE, Schneider JW, Chen S, Li L, Sawaya MR, Eisenberg D, Tycko R, and McKnight SL. (2012). Cell-free formation of RNA granules: Low complexity sequence domains form dynamic fibers within hydrogels. Cell. 149, 753-767.
Kwon I, Xiang S, Kato M, Wu L, Theodoropoulos P, Wang T, Kim J, Yun J, Xie Y, and McKnight SL. (2014). Poly-dipeptides encoded by the C9orf72 repeats bind nucleoli, impede RNA biogenesis, and kill cells. Science. 345, 1139-1145.
Xiang S, Kato M, Wu L, Lin Y, Ding M, Zhang Y, Yu Y, and McKnight SL. (2015). The LC domain of hnRNPA2 adopts similar conformations in hydrogel polymers, liquid-like droplets, and nuclei. Cell. 163, 829-839.
Murray DT, Kato M, Lin Y, Thurber KR, Hung I, and McKnight SL, and Tycko R. (2017). Structure of FUS protein fibrils and its relevance to self-assembly and phase separation of low-complexity domains. Cell. 171, 615-627.
Zhou X, Lin Y, Kato M, Mori E, Liszczak G, Sutherland L, Sysoev VO, Murray DT, Tycko R, and McKnight SL. (2021). Transiently structured head domains control intermediate filament assembly. Proc. Natl. Acad. Sci. USA. 118. doi: 10.1073/pnas.2022121118.
Zhou X, Sumrow L, Tashiro K, Sutherland L, Liu D, Qin T, Kato M, Liszczak G, and McKnight SL. (2022). Mutations linked to neurological disease enhance self-association of low-complexity protein sequences. Science. 377, 1-22. doi.org/10.1126/science.abn5582.
Kato M, Zhou X, and McKnight SL. (2022). How do protein domains of low sequence complexity work? RNA. 28, 3-15.
Award Presentation: James Rothman
Our genome specifies tens of thousands of proteins, each with a different job to do. To avoid mix-ups, proteins with related jobs are collected together into groups called organelles where they cooperate to achieve major tasks, like generating energy from food, or making new proteins, or storing information.
These organelles are easily seen with an electron microscope because they are enclosed by a conspicuous membrane. Among them is the nucleus, which contains the cell’s chromosomes and the proteins that copy them. Like other organelles, the proteins grouped inside the nucleus are targeted individually and literally placed inside one at a time.
That is how we understood cell organization for over half a century. But, especially in the last decade, we have come to realize there are likely hundreds of other groupings that were initially invisible to the electron microscope because they lacked outer membranes, a kind of cellular “dark matter” that mostly eluded us. These proteins are not transported one at a time into a pre-existing organelle – rather they group-up all at once and by themselves, sticking loosely to each other and separating away from the cell as a whole.
Just as dark matter promises to revolutionize our understanding of the physical world, these shape-shifting conglomerates have changed the way we think about cell organization and are shedding new light on neurodegeneration and cancer.
Today’s Basic Science Laureates discovered the basic principles involved. The “dark matter” proteins have a special “super-power” that enables them to group-up automatically, entirely on their own, whenever and wherever they are needed.
Acceptance remarks
Acceptance Remarks: Dirk Görlich
I was instructed to not thank anyone in my acceptance remarks. So, let me share some moments on the road that led me here.
Ever since I was a kid, I was captivated by chemistry and impressed by the early organic chemists who got structures right without any high-tech instruments, and in particularly by Kekulé, who put the six carbons and six hydrogens of benzene into a meaningful chemical structure. The story goes that he had a dream of the six carbon atoms dancing, holding their hands and forming a circle – the six-membered aromatic ring that we all know today.
We had our Kekulé moment as well, namely when explaining how nuclear pores form a permeability barrier from disordered FG repeat domains. The solution was not immediately obvious but simple in the end. These very long linear polymers have to touch each other to form sieve-like meshes. To do this, they don’t just use just two hands, they use many sticky spots. There is now another connection to Kekulé. These sticky spots are made by the amino acid phenylalanine that occurs at every ~12th position of the sequence and contains a benzene ring. These benzene rings like touching each other much more than floating alone through the water. This “touching” creates the new material we call the FG phase, or more generally, a biomolecular condensate.
It took us several years to establish the biochemistry for reconstituting such barrier material in a test tube. Eventually, we put it on the stage of a confocal laser-scanning microscope and could watch it repelling normal macromolecules, while sucking-in the transporters that carry cargo through nuclear pores. Amazingly, this material reached a sorting fidelity of 10 000: 1. It recapitulated the very high sorting speed of nuclear pores that operate near the diffusion limit. Like real nuclear pores, the barrier material could transport cargoes of very different size and shape – provided the cognate transporters were present.
Even though these observations were very clear and intuitive, the nuclear transport field was remarkably reluctant to accept that nuclear pores use something as simple as a condensed phase for traffic control. In the end, it helped that similar condensates were found throughout cells. Scientific discovery is rarely a straight line. There will always be headwinds—those who question your ideas or resist new ways of thinking. The key is to design experiments not to please expectations but to put ideas at the strictest possible test and find satisfaction not just in the destination, but in the journey of discovering something new.
Acceptance Remarks: Steven McKnight
In 1996 UTSWMC appointed me as chair of the Biochemistry Department. I was not a chemist in 1996, and it is questionable as to whether I qualify as being called a chemist today.
Despite a profound lack of qualifications, I chose to invest in synthetic chemistry as a path towards reinvigoration of our Biochemistry Department. There were two reasons for choosing chemistry. First, we had no chemists working in the department. If we wanted to retain the name of biochemistry, we had to hire chemistry faculty. Second, the 800-pound gorilla of biomedical research in the 1990’s was genomics. Since there was no chance for our department to be competitive in the field of genomics, we chose a direction as orthogonal to genomics as we could possibly conceive.
Before joining UT Southwestern I had studied gene regulation. I naively thought that it might be possible to discover chemicals capable of modulating transcription. Perhaps these sorts of chemicals could represent starting points for new medicines. A combination of hardheadedness and the scientific talents of colleagues and trainees led us to discover just that – a chemical inhibitor of the HIF2α transcription factor. This prompted the founding of a biotech company that optimized the HIF2α inhibitor and performed the clinical trials required for FDA approval of its use in the treatment of kidney cancer.
The HIF2α story represents the intended consequence of our decision to bet on chemistry. The unintended consequences of this decision ended up being more profound. Chemistry became the tail that wagged the dog of my research program for the remainder of my career.
I stand here today because Eric Olson used our high throughput drug screening facility to discover a chemical that causes stem cells to differentiate into heart muscle cells. Joe Ready put a biotin group on Eric’s chemical to help discover its mode of action. Whereas those efforts failed, students working with me found that the biotinylated derivative of Eric’s chemical selectively precipitates virtually all RNA binding proteins from cell lysates. In simple terms, this chemical allows for the test tube reconstitution of RNA granules. By chasing down this outrageously irrelevant observation, we came to realize how protein domains of low sequence complexity work.
The discomfort of a foreign discipline ensured that I would not spend my last decades in the timid decay of my familiarity with gene expression. It was the unintended consequence of choosing chemistry that enabled me to stumble over the magic of serendipity, and it is serendipity that has become a rate-limiting ingredient of modern biomedical research.
2025 Basic Award video
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