Zooming in on the cell biology of disease

Today’s cell biology could be considered a fusion of disciplines that blends advanced genetics, molecular biology, biochemistry, and engineering to answer fundamental as well as medically relevant scientific questions. Accordingly, our understanding of diseases is greatly aided by an existing vast knowledge base of fundamental cell biology. Gunter Blobel captured this concept when he said, “the tremendous acquisition of basic knowledge will allow a much more rational treatment of cancer, viral infection, degenerative disease and mental disease.” In other words, without cell biology can we truly understand, prevent, or effectively treat a disease?

R. M. Perera     

George E. Palade, Cell Biology and The JCB

On October 7, 2008, the world lost one of the most influential scientists of the 20th century, and modern cell biology lost its founder. George E. Palade, recipient of the Nobel Prize in 1974 for his work that established our basic understanding of cellular organization, died at the age of 95 after a long illness.     

A Nobel Prize for membrane traffic: Vesicles find their journey’s end

Cell biologists everywhere rejoiced when this year’s Nobel Prize in Physiology or Medicine was awarded to James Rothman, Randy Schekman, and Thomas Südhof for their contributions to uncovering the mechanisms governing vesicular transport. In this article, we highlight their achievements and also pay tribute to the pioneering scientists before them who set the stage for their remarkable discoveries.In 1974, nearly 40 years ago, the Nobel Prize in Physiology or Medicine was awarded to George E. Palade, Albert Claude, and Christian de Duve for work that effectively established a new field, cell biology. Collectively, the efforts of these three pioneers not only defined the essential features of cells but also how to study them. Correlating morphological observations by electron microscopy with biochemical analysis enabled not only the identification of nearly every major organelle in the eukaryotic cell (although endosomes were missed at that time) but also what their respective functions were. Palade’s efforts demonstrated the now-canonical pathway of protein secretion: synthesis in the endoplasmic reticulum (ER), oligosaccharide processing in the Golgi complex, concentration in secretory granules, and release at the plasma membrane. Palade understood implicitly that the ER, Golgi, secretory granules, and plasma membrane had to be interconnected by a series of vesicular carriers that carried cargo from one station to the next—dissociative transport. He also appreciated that the process had to be regulated if compartment specificity was to be maintained. The need for specificity defined the next major conceptual challenges: how do proteins intended for secretion traverse the compartments of the secretory pathway, how are transport vesicles formed, how do vesicles recognize their appropriate destinations, how does fusion occur after the appropriate destination is reached, and, finally, how are the components from the originating compartment returned or recycled to their sites of origin after fusion with the destination compartment? Palade may have framed these problems, but it was left to the next generation of cell biologists to solve them.This year’s Nobel Prize in Physiology or Medicine awarded to James Rothman, Randy Schekman, and Thomas Südof recognizes a truly remarkable body of work that provides superb conceptual clarity and mechanistic insight into virtually all of the issues defined by Palade and colleagues. To a large extent, the award also provides a satisfying degree of recognition to the large community of scientists who established the field of “molecular” cell biology. But it was the intellectual leadership, passion, and courage provided by this year’s awardees (Figs. 1 and ​and2)2) that played a major role in driving the spectacular advances of the past three decades. Particularly in the case of Rothman and Schekman, the scientific dynamic they helped to generate gave the field focus and excitement, from which came great things. The elegance of their experiments together with the exceptionally clear and simple logic that they presented in their papers moved the field ahead quickly and drew many new converts into membrane trafficking.Open in a separate windowFigure 1.Randy Schekman and James Rothman (center) with many of their former trainees at the American Society for Biochemistry and Molecular Biology meeting on “Biochemistry of Membrane Traffic: Secretory and Endocytic Pathways,” October 2010.PHOTOGRAPH COURTESY OF THE AMERICAN SOCIETY FOR BIOCHEMISTRY AND MOLECULAR BIOLOGYOpen in a separate windowFigure 2.Thomas Südhof (top row, center) and his laboratory circa 1993.PHOTOGRAPH COURTESY OF THOMAS SÜDHOFThe first foray into a mechanistic, molecular approach to the cell biological problems defined by Palade was really due to the work of Günter Blobel and his colleagues Peter Walter and Bernhard Dobberstein working at The Rockefeller University. These investigators devised a complex but elegant approach enabling the cell-free reconstitution of the first step of secretion, namely the insertion of newly synthesized proteins into and across the ER membrane. Combined with conventional cold-room biochemistry, Blobel and others were able to provide a detailed understanding of the biochemistry of protein translocation. Blobel was duly awarded the Nobel Prize in Physiology or Medicine for his work in 1999. Influenced by Blobel and also Arthur Kornberg, then chair of the Biochemistry Department at Stanford, Jim Rothman (who was a young faculty member at Stanford in the early 1980s) initiated his courageous effort aimed to reconstitute subsequent steps, namely the transport of secretory and membrane protein cargo to and through the Golgi complex. As is often the case with innovative work that pushes the limits of knowledge, Rothman’s interpretations were on occasion controversial, but there was absolutely no controversy regarding the importance of the various components he and his team identified. These components included soluble factors needed for vesicle formation in the Golgi as well as for vesicle fusion, most notably the COPI coat protein complex, NSF (NEM-sensitive factor) and SNAP (soluble NSF attachment protein). With Richard Scheller, Rothman recognized that the synaptic vesicle–associated proteins cloned and purified by Scheller represented both the docking sites for NSF and SNAP and a key component of the mechanism whereby vesicles recognized and even fused with each other. Indeed, the SNAREs (as these proteins are now called) clearly comprise the core fusion machinery that underlies virtually all membrane fusion events in the cell. SNAREs form a family of proteins that are organelle specific, helping to ensure the specificity of membrane traffic as well as the biochemical and functional identity of individual membrane compartments.If Rothman’s work began as a quintessential biochemical approach, Randy Schekman’s started at the other end of the spectrum: genetics. Again with great courage, Schekman decided to use the yeast Saccharomyces cerevisiae as a genetically tractable eukaryote to dissect the steps and various components associated with the secretory pathway. At the time, few thought that yeast cells were capable of higher-order processes such as secretion or that their activities had anything to do with mechanisms in animal cells. Yet Schekman and his then graduate student Peter Novick designed a deceptively simple screen to identify secretory (or “sec”) mutants. Their approach was to look for cells that could not secrete by reasoning that continued synthesis of secretory cargo would render the mutant cells more dense. The approach worked, and literally dozens of mutants were discovered, a large number of which could be shown to generate intriguing phenotypes and to control key steps in the secretory, or sometimes even the endocytic, pathway. Although the original sec screens done by Schekman and colleagues did not immediately turn up the SNARE proteins, they did reveal the presence of small Ras-related monomeric GTPases of the Rab family that helped enforce the specificity of vesicle interactions. They also uncovered cytoplasmic coat proteins (COPII) and complex cytosolic “tethers” that serve to gather vesicles at their targets before the final fusion step. When an increasing number of sec mutants began to overlap with components identified by Rothman’s independent biochemical purifications of components required for fusion or vesicle budding, it was clear that both groups (and indeed the field) were on the right track and the transport machinery was universal. Through whatever controversies bubbled up over the years, this basic fact remained unchallenged. Schekman too moved toward the same type of functional biochemical analysis championed by Rothman, and the circle was completed.Focused on one of the key problems in neurobiology, Thomas Südhof’s efforts may appear less general but are no less important. The synapse represented a special case in the area of membrane traffic since the realization that neuronal transmission reflected the release of quanta of neurotransmitters due to the action potential–triggered fusion of synaptic vesicles with the presynaptic plasma membrane. The work of Cesare Montecucco and colleagues on bacterial toxins provided an important insight, namely that synaptic vesicle release can be blocked by certain bacterial toxins (e.g., botulinum toxin) that act as specific SNARE proteases. Scheller and Rothman had shown that the SNAREs comprised the basic unit of the fusion machinery, but this insight alone did not explain how secretion in the synapse was coupled so tightly to electrical activity. Thomas Südhof’s remarkable body of work, although not growing out of the molecular cell biology community as much as the neuroscience community, provided the conceptual answer: the synaptotagmins. These proteins were found to associate with SNAREs and serve as Ca²⁺-sensing triggers that temporally linked synaptic vesicle transmission to individual neuronal impulses. In addition, Südhof and colleagues discovered Munc18 in the mouse, which corresponds to the yeast Sec1 protein, and demonstrated that it interacts with the SNARE complex, revealing that Munc18 as well as other members of the Sec1/Munc18-like protein family function as part of the vesicle fusion machinery.Collectively, these are remarkable achievements that provide conceptual and mechanistic understanding of basic cellular processes at the most fundamental level. It is certainly the case that others, for example Scheller and Novick mentioned here, might just as easily have been included in this award. Regrettably only three are permitted, and there can be no doubt but that the three selected are entirely deserving given not only the nature of their findings but also the scientific leadership they contributed in a myriad of intangible ways to the incredible progress we have witnessed in the post-Palade era of cell biology.We congratulate our colleagues and friends Jim, Randy, and Thomas for this well-deserved honor. Mazal tov!     

Nucleoporins’ exclusive amino acid sequence features regulate their transient interaction with and selectivity of cargo complexes in the nuclear pore

Nucleocytoplasmic traffic of nucleic acids and proteins across the nuclear envelop via the nuclear pore complexes (NPCs) is vital for eukaryotic cells. NPCs screen transported macromolecules based on their morphology and surface chemistry. This selective nature of the NPC-mediated traffic is essential for regulating the fundamental functions of the nucleus, such as gene regulation, protein synthesis, and mechanotransduction. Despite the fundamental role of the NPC in cell and nuclear biology, the detailed mechanisms underlying how the NPC works have remained largely unknown. The critical components of NPCs enabling their selective barrier function are the natively unfolded phenylalanine- and glycine-rich proteins called “FG-nucleoporins” (FG Nups). These intrinsically disordered proteins are tethered to the inner wall of the NPC, and together form a highly dynamic polymeric meshwork whose physicochemical conformation has been the subject of intense debate. We observed that specific sequence features (called largest positive like-charge regions, or lpLCRs), characterized by extended subsequences that only possess positively charged amino acids, significantly affect the conformation of FG Nups inside the NPC. Here we investigate how the presence of lpLCRs affects the interactions between FG Nups and their interactions with the cargo complex. We combine coarse-grained molecular dynamics simulations with time-resolved force distribution analysis to disordered proteins to explore the behavior of the system. Our results suggest that the number of charged residues in the lpLCR domain directly governs the average distance between Phe residues and the intensity of interaction between them. As a result, the number of charged residues within lpLCR determines the balance between the hydrophobic interaction and the electrostatic repulsion and governs how dense and disordered the hydrophobic network formed by FG Nups is. Moreover, changing the number of charged residues in an lpLCR domain can interfere with ultrafast and transient interactions between FG Nups and the cargo complex.     

Charting the secretory pathway in a simple eukaryote

George Palade, a founding father of cell biology and of the American Society for Cell Biology (ASCB), established the ultrastructural framework for an analysis of how proteins are secreted and membranes are assembled in eukaryotic cells. His vision inspired a generation of investigators to probe the molecular mechanisms of protein transport. My laboratory has dissected these pathways with complementary genetic and biochemical approaches. Peter Novick, one of my first graduate students, isolated secretion mutants of Saccharomyces cerevisiae, and through cytological analysis of single and double mutants and molecular cloning of the corresponding SEC genes, we established that yeast cells use a secretory pathway fundamentally conserved in all eukaryotes. A biochemical reaction that recapitulates the first half of the secretory pathway was used to characterize Sec proteins that comprise the polypeptide translocation channel in the endoplasmic reticulum (ER) membrane (Sec61) and the cytoplasmic coat protein complex (COPII) that captures cargo proteins into transport vesicles that bud from the ER.     

A biosensor of protein foldedness identifies increased “holdase” activity of chaperones in the nucleus following increased cytosolic protein aggregation

Chaperones and other quality control machinery guard proteins from inappropriate aggregation, which is a hallmark of neurodegenerative diseases. However, how the systems that regulate the “foldedness” of the proteome remain buffered under stress conditions and in different cellular compartments remains incompletely understood. In this study, we applied a FRET-based strategy to explore how well quality control machinery protects against the misfolding and aggregation of “bait” biosensor proteins, made from the prokaryotic ribonuclease barnase, in the nucleus and cytosol of human embryonic kidney 293T cells. We found that those barnase biosensors were prone to misfolding, were less engaged by quality control machinery, and more prone to inappropriate aggregation in the nucleus as compared with the cytosol, and that these effects could be regulated by chaperone Hsp70-related machinery. Furthermore, aggregation of mutant huntingtin exon 1 protein (Httex1) in the cytosol appeared to outcompete and thus prevented the engagement of quality control machinery with the biosensor in the cytosol. This effect correlated with reduced levels of DNAJB1 and HSPA1A chaperones in the cell outside those sequestered to the aggregates, particularly in the nucleus. Unexpectedly, we found Httex1 aggregation also increased the apparent engagement of the barnase biosensor with quality control machinery in the nucleus suggesting an independent implementation of “holdase” activity of chaperones other than DNAJB1 and HSPA1A. Collectively, these results suggest that proteostasis stress can trigger a rebalancing of chaperone abundance in different subcellular compartments through a dynamic network involving different chaperone–client interactions.     

Moving beyond molecular mechanisms

A major goal in cell biology is to bridge the gap in our understanding of how molecular mechanisms contribute to cell and organismal physiology. Approaches well established in the physical sciences could be instrumental in achieving this goal. A better integration of the physical sciences with cell biology will therefore be an important step in our quest to decipher how cells work together to construct a living organism.Over the past 60 years, the field of cell biology has been firmly rooted in understanding the molecular basis of complex cellular processes including genome replication, migration, metabolism, and adhesion. This progress has been enabled by advances in molecular biology, biochemistry, physical chemistry, single-molecule physics, and microscopy. Bringing together these disciplines has been successful in identifying the molecular composition of macromolecular machines, characterizing the structure and physical properties of single proteins within cells, reconstituting complex macromolecular machinery in vitro, and imaging the dynamics and function of these machines in vivo.Despite this amazing progress, a major challenge facing cell biology is understanding how the chemical and physical properties of molecular machinery come together to guide cell processes. How do varied physical and chemical signals in the environment determine whether a cell survives, proliferates, or migrates? What circuitry allows for a complex body plan to be constructed out of a single-celled embryo? The signals in the environment are noisy, with fluctuations in both time and space. Moreover, as anyone who has tried to characterize cells is aware, cell phenotypes are variable both across individual cells and within a single cell over time. In the presence of all this noise, cells execute some processes exceedingly reliably (e.g., DNA segregation in cell division). Others, such as the determination of protrusive activity in a migrating cell, appear to be more variable. How does this complex network of stochastic chemical and mechanical machinery enable robust and complex decision making at the cell scale?The answers to these questions require knowledge of cell structure at the scale between single molecules and whole cells (Fig. 1). This intermediate, or mesoscopic, length scale has different names depending on who you ask. You can think of it as a “system” or interconnected network of biochemical interactions that provide a logic circuit as to how cells process a signal to decide on an output. It can be a subcellular machine consisting of a collection of macromolecules designed to work together for a desired mechanical output, such as cargo transport, DNA segregation, or cell movement. There is a significant gap in our understanding at this scale. To make an analogy between a cell and a car: most of us have a good understanding of the car’s component materials (e.g., rubber, metal), and in some cases we understand the individual machines that make up parts of the whole (e.g., the engine, transmission). However, we do not have a good understanding of the essential control parameters of the machines or how these are wired together to form productive, more complex machinery (e.g., creating the forward, backward, and turning motions). Understanding the control parameters that regulate macromolecular assemblies, and how these are wired together to enable complex cell outputs, represents an exciting frontier in cell biology.Open in a separate windowFigure 1.The scales of cell biology. Shown are images illustrating the range of scales in cell biology. At the smallest (∼10⁻⁹ m) is that of molecules represented by the structure of G-actin (left; reproduced from Paavilainen et al. 2008. J. Cell Biol. http://dx.doi.org/10.1083/jcb.200803100) and the largest (10⁻⁵ to 10⁻⁴ meters) is that of cell physiology, represented by a migrating fibroblast with a labeled actin cytoskeleton (right; image courtesy of Patrick Oakes). In between these length scales reside: macromolecular assemblies (10⁻⁸ to 10⁻⁷ m) of individual proteins, represented by a schematic of an Arp2/3-mediated F-actin branch (second from the left); and organelles (10⁻⁷ to 10⁻⁵ m), such as lamellipodia (third from the left), which are formed by the integration of macromolecular assemblies into a mechanochemical machine depicted as a pathway diagram. At the next level are organelle systems (10⁻⁴ to 10⁻⁵ m) that integrate organelles together for a specific aspect of cell physiology, represented by a fluorescent image of actin overlaid with vectors of actin flow at the leading edge that result from the coordination of numerous regulatory organelles across the cell (second from the right; reproduced from Thievessen et al. 2013. J. Cell Biol. http://dx.doi.org/10.1083/jcb.201303129). Understanding the processes at this intermediate scale will greatly aid in our knowledge of how molecules construct living cells.Many areas of the physical sciences have been devoted to studying how collections of objects work together to construct a material or machine. In this construction, new properties emerge that could not be predicted or understood by studies of objects in isolation. For instance, electrical engineers need to know how circuit elements are connected in order to predict the circuit response. Or, in condensed matter physics, interactions between atoms and/or molecules result in properties such as elasticity or viscosity. In these areas of science, it is well appreciated that knowledge of individual components (in isolation) cannot predict the output of the entire system. By analogy, this would imply that understanding the molecular components of a cell, which has been the gold standard of cell biology, is insufficient. As cell biology starts to address questions wherein cells are thought of as “systems,” “materials,” or “machines,” there are numerous challenges that can be informed by approaches that have proven successful in the studies of materials and machines in the physical world.

Developing a common community

Cell biology is an inherently multidisciplinary science, requiring approaches from genetics, chemistry, physics, applied mathematics, and engineering. While biochemical and genetic approaches have been successfully integrated into the field, other disciplines require more effort. Physical scientists that join the field of cell biology retain the training and language from their physical discipline, which has been specialized for specific purposes. Applied mathematicians, condensed matter physicists, and mechanical engineers all have unique perspectives on how to model complex biological phenomena (Fig. 2). This has led to the development of parallel theoretical and experimental approaches for modeling cell biological phenomena that are difficult to directly compare or rigorously test. A challenge for the future is to develop a community of researchers that will integrate these diverse physical approaches to identify strengths, resolve differences, and determine the best approaches for modeling cell behaviors.Open in a separate windowFigure 2.The integration of physical sciences with cell biology. A flow chart showing examples of how various disciplines from the physical sciences (bottom) have optimized a variety of theoretical/modeling tools (left) as well as experimental techniques (right) that have been applied to cell biological problems. However, these experimental and theoretical tools have been optimized for their home disciplines. A current challenge is to systematically have them benchmarked against each other and identify their weaknesses and strengths before using them to provide a new framework optimized for mesoscale cell biology.

Precision in language

One of the simplest solutions to implement is to develop a consistent and precise language to describe measurements or ideas. In my field, which centers on how mechanical forces are sensed and generated by cells, terms like “mechanosensing” or even “stiffness sensing” are used without precision, resulting in confusion of what is known versus just “thought to be true.” Precision of language is essential for standardizing experimental protocols and measurements and in being able to clearly communicate conclusions and ideas.

Construction and validation of physical methods

One historical role of physical scientists in biology has been the introduction of new experimental and analytical tools. Some of these tools, such as microscopy and scattering techniques, have been developed extensively. However, in other cases, the nature of measurements require small apparatuses that can be difficult to replicate or operate (magnetic tweezers are a notorious example), making it difficult for other laboratories to build upon this knowledge. Similar issues arise in analysis and methods. It is extremely important for these methods to be used and validated by different laboratories to confirm results independently and by many individuals so that the language used to describe physical concepts and results can be made more precise. Being able to directly compare two different measurement techniques so that the same parameters can be used is essential for resolving discrepancies.Even though the goal is to understand cell physiology, model testing will require physical characterization that may not immediately inform a biological process. To use an analogous example: the work in basic materials science of magnetism that needed to be performed before we could construct and build computer hard drives. It is my hope that the cell biology community will remain interested in these advances in characterization of biological materials and systems, as they are crucial to uncovering synergies that are not currently apparent.

Feedback between modeling and experiments

In the physical sciences, research has evolved so that individuals typically focus on either theory or experimentation. Of course, each of these can be further subdivided into analytical theory versus computer modeling, as well as sample preparation versus characterization. This specialization has emerged as both the questions and fields themselves become more mature. It also has led to a vigorous feedback between theoretical prediction, experimental measurement, and new materials development. To be useful, models need to be falsifiable. There is increasing evidence that many of the models used in biology are over-parameterized and, consequently, difficult (or impossible) to falsify. That is, when parameters are assigned with molecular-level details, the number of parameters quickly becomes large. In these scenarios, changes in the parameter value have little effect on the model predictions and make it difficult to verify the accuracy of the model (for more details, see http://www.lassp.cornell.edu/sethna/Sloppy/). Identifying order parameters that encompass the physical quantities or metrics (e.g., elastic modulus, organelle transport) that make up many of the molecular details is essential for developing models with fewer control parameters. Such order parameters will provide crucial insight into understanding regulation of the individual macromolecular machinery.The word mechanism in cell biology typically refers to a molecular mechanism that is explored rigorously by genetic and biochemical testing. Understanding the physical mechanism requires both identification of the parameters controlling a system and then elucidation of the regulation of parameter values. Thus, seldom does a single molecular mechanism tie directly into a physical parameter. Moreover, understanding how molecular interactions give rise to a single physical parameter is not straightforward, and may require years of work. It is quite natural to apply models and approaches that we have used to engineer machines, such as the flow of decision making in electrical circuits or mechanic designs. However, cells are working under different sets of constraints, and a future challenge of understanding cellular machines is that completely different design principles may be used.Establishing a culture that encourages dynamic feedback between theory, experimentation, and physiology is crucial to advancing the integration of physical sciences with cell biology. A potentially very exciting possibility is that understanding the physical mechanisms controlling biological machines will enable a completely new set of design principles that provide insight into how living cells are able to respond and adapt to highly variable environments. This will enable understanding of how these states change during disease progression and the capability of engineering biological cells to maintain a healthy phenotype.     

Sphingolipids: regulators of crosstalk between apoptosis and autophagy

Apoptosis and autophagy are two evolutionarily conserved processes that maintain homeostasis during stress. Although the two pathways utilize fundamentally distinct machinery, apoptosis and autophagy are highly interconnected and share many key regulators. The crosstalk between apoptosis and autophagy is complex, as autophagy can function to promote cell survival or cell death under various cellular conditions. The molecular mechanisms of crosstalk are beginning to be elucidated and have critical implications for the treatment of various diseases, such as cancer. Sphingolipids are a class of bioactive lipids that mediate many key cellular processes, including apoptosis and autophagy. By targeting several of the shared regulators, sphingolipid metabolites differentially regulate the induction of apoptosis and autophagy. Importantly, individual sphingolipid species appear to “switch” autophagy toward cell survival (e.g., sphingosine-1-phosphate) or cell death (e.g., ceramide, gangliosides). This review assesses the current understanding of sphingolipid-induced apoptosis and autophagy to address how sphingolipids mediate the “switch” between the cell survival and cell death. As sphingolipid metabolism is frequently dysregulated in cancer, sphingolipid-modulating agents, or sphingomimetics, have emerged as a novel chemotherapeutic strategy. Ultimately, a greater understanding of sphingolipid-mediated crosstalk between apoptosis and autophagy may be critical for enhancing the chemotherapeutic efficacy of these agents.     

Alternate histories of cytokinesis: lessons from the trypanosomatids

Popular culture has recently produced several “alternate histories” that describe worlds where key historical events had different outcomes. Beyond entertainment, asking “could this have happened a different way?” and “what would the consequences be?” are valuable approaches for exploring molecular mechanisms in many areas of research, including cell biology. Analogous to alternate histories, studying how the evolutionary trajectories of related organisms have been selected to provide a range of outcomes can tell us about the plasticity and potential contained within the genome of the ancestral cell. Among eukaryotes, a group of model organisms has been employed with great success to identify a core, conserved framework of proteins that segregate the duplicated cellular organelles into two daughter cells during cell division, a process known as cytokinesis. However, these organisms provide relatively sparse sampling across the broad evolutionary distances that exist, which has limited our understanding of the true potential of the ancestral eukaryotic toolkit. Recent work on the trypanosomatids, a group of eukaryotic parasites, exemplifies alternate historical routes for cytokinesis that illustrate the range of eukaryotic diversity, especially among unicellular organisms.     

An updated SYSCILIA gold standard (SCGSv2) of known ciliary genes,revealing the vast progress that has been made in the cilia research field

Cilia are microtubule-based organelles with important functions in motility and sensation. They contribute to a broad spectrum of developmental disorders called ciliopathies and have recently been linked to common conditions such as cancers and congenital heart disease. There has been increasing interest in the biology of cilia and their contribution to disease over the past two decades. In 2013 we published a “Gold Standard” list of genes confirmed to be associated with cilia. This was published as part of the SYSCILIA consortium for systems biology study dissecting the contribution of cilia to human health and disease, and was named the Syscilia Gold Standard (SCGS). Since this publication, interest in cilia and understanding of their functions have continued to grow, and we now present an updated SCGS version 2. This includes an additional 383 genes, more than doubling the size of SCGSv1. We use this dataset to conduct a review of advances in understanding of cilia biology 2013– 2021 and offer perspectives on the future of cilia research. We hope that this continues to be a useful resource for the cilia community.     

Bacterial evolution during human infection: Adapt and live or adapt and die

Microbes are constantly evolving. Laboratory studies of bacterial evolution increase our understanding of evolutionary dynamics, identify adaptive changes, and answer important questions that impact human health. During bacterial infections in humans, however, the evolutionary parameters acting on infecting populations are likely to be much more complex than those that can be tested in the laboratory. Nonetheless, human infections can be thought of as naturally occurring in vivo bacterial evolution experiments, which can teach us about antibiotic resistance, pathogenesis, and transmission. Here, we review recent advances in the study of within-host bacterial evolution during human infection and discuss practical considerations for conducting such studies. We focus on 2 possible outcomes for de novo adaptive mutations, which we have termed “adapt-and-live” and “adapt-and-die.” In the adapt-and-live scenario, a mutation is long lived, enabling its transmission on to other individuals, or the establishment of chronic infection. In the adapt-and-die scenario, a mutation is rapidly extinguished, either because it carries a substantial fitness cost, it arises within tissues that block transmission to new hosts, it is outcompeted by more fit clones, or the infection resolves. Adapt-and-die mutations can provide rich information about selection pressures in vivo, yet they can easily elude detection because they are short lived, may be more difficult to sample, or could be maladaptive in the long term. Understanding how bacteria adapt under each of these scenarios can reveal new insights about the basic biology of pathogenic microbes and could aid in the design of new translational approaches to combat bacterial infections.     

Single Cell Genomics of the Brain: Focus on Neuronal Diversity and Neuropsychiatric Diseases

Single cell genomics has made increasingly significant contributions to our understanding of the role that somatic genome variations play in human neuronal diversity and brain diseases. Studying intercellular genome and epigenome variations has provided new clues to the delineation of molecular mechanisms that regulate development, function and plasticity of the human central nervous system (CNS). It has been shown that changes of genomic content and epigenetic profiling at single cell level are involved in the pathogenesis of neuropsychiatric diseases (schizophrenia, mental retardation (intellectual/leaning disability), autism, Alzheimer’s disease etc.). Additionally, several brain diseases were found to be associated with genome and chromosome instability (copy number variations, aneuploidy) variably affecting cell populations of the human CNS. The present review focuses on the latest advances of single cell genomics, which have led to a better understanding of molecular mechanisms of neuronal diversity and neuropsychiatric diseases, in the light of dynamically developing fields of systems biology and “omics”.     

Many chronological aging clocks can be found throughout the epigenome: Implications for quantifying biological aging

Epigenetic alterations are a hallmark of aging and age‐related diseases. Computational models using DNA methylation data can create “epigenetic clocks” which are proposed to reflect “biological” aging. Thus, it is important to understand the relationship between predictive clock sites and aging biology. To do this, we examined over 450,000 methylation sites from 9,699 samples. We found ~20% of the measured genomic cytosines can be used to make many different epigenetic clocks whose age prediction performance surpasses that of telomere length. Of these predictive sites, the average methylation change over a lifetime was small (~1.5%) and these sites were under‐represented in canonical regions of epigenetic regulation. There was only a weak association between “accelerated” epigenetic aging and disease. We also compare tissue‐specific and pan‐tissue clock performance. This is critical to applying clocks both to new sample sets in basic research, as well as understanding if clinically available tissues will be feasible samples to evaluate “epigenetic aging” in unavailable tissues (e.g., brain). Despite the reproducible and accurate age predictions from DNA methylation data, these findings suggest they may have limited utility as currently designed in understanding the molecular biology of aging and may not be suitable as surrogate endpoints in studies of anti‐aging interventions. Purpose‐built clocks for specific tissues age ranges or phenotypes may perform better for their specific purpose. However, if purpose‐built clocks are necessary for meaningful predictions, then the utility of clocks and their application in the field needs to be considered in that context.     

Onward from the cradle

This essay records a voyage of discovery from the “cradle of cell biology” to the present, focused on the biology of the oldest known cell organelle, the cilium. In the “romper room” of cilia and microtubule (MT) biology, the sliding MT hypothesis of ciliary motility was born. From the “summer of love,” students and colleagues joined the journey to test switch-point mechanisms of motility. In the new century, interest in nonmotile (primary) cilia, never lost from the cradle, was rekindled, leading to discoveries relating ciliogenesis to autophagy and hypotheses of how molecules cross ciliary necklace barriers for cell signaling.     

Coordination between cell proliferation and apoptosis after DNA damage in Drosophila

Exposure to genotoxic stress promotes cell cycle arrest and DNA repair or apoptosis. These “life” or “death” cell fate decisions often rely on the activity of the tumor suppressor gene p53. Therefore, the precise regulation of p53 is essential to maintain tissue homeostasis and to prevent cancer development. However, how cell cycle progression has an impact on p53 cell fate decision-making is mostly unknown. In this work, we demonstrate that Drosophila p53 proapoptotic activity can be impacted by the G2/M kinase Cdk1. We find that cell cycle arrested or endocycle-induced cells are refractory to ionizing radiation-induced apoptosis. We show that p53 binding to the regulatory elements of the proapoptotic genes and its ability to activate their expression is compromised in experimentally arrested cells. Our results indicate that p53 genetically and physically interacts with Cdk1 and that p53 proapoptotic role is regulated by the cell cycle status of the cell. We propose a model in which cell cycle progression and p53 proapoptotic activity are molecularly connected to coordinate the appropriate response after DNA damage.Subject terms: Cell biology, Development, Gene regulation, Molecular biology     

High-throughput Screening in Embryonic Stem Cell-derived Neurons Identifies Potentiators of α-Amino-3-hydroxyl-5-methyl-4-isoxazolepropionate-type Glutamate Receptors

Stem cell biology offers advantages to investigators seeking to identify new therapeutic molecules. Specifically, stem cells are genetically stable, scalable for molecular screening, and function in cellular assays for drug efficacy and safety. A key hurdle for drug discoverers of central nervous system disease is a lack of high quality neuronal cells. In the central nervous system, α-amino-3-hydroxyl-5-methyl-4-isoxazolepropionate (AMPA) subtype glutamate receptors mediate the vast majority of excitatory neurotransmissions. Embryonic stem (ES) cell protocols were developed to differentiate into neuronal subtypes that express AMPA receptors and were pharmacologically responsive to standard compounds for AMPA potentiation. Therefore, we hypothesized that stem cell-derived neurons should be predictive in high-throughput screens (HTSs). Here, we describe a murine ES cell-based HTS of a 2.4 × 10⁶ compound library, the identification of novel chemical “hits” for AMPA potentiation, structure function relationship of compounds and receptors, and validation of chemical leads in secondary assays using human ES cell-derived neurons. This reporting of murine ES cell derivatives being formatted to deliver HTS of greater than 10⁶ compounds for a specific drug target conclusively demonstrates a new application for stem cells in drug discovery. In the future new molecular entities may be screened directly in human ES or induced pluripotent stem cell derivatives.