Origin and prediction of free-solution interaction studies performed label-free
Darryl J. Bornhop, Michael N. Kammer, Amanda Kussrow, Robert A. Flowers II, and Jens Meiler
Chemical and biomedical sciences depend heavily on interaction assays, particularly those providing structural insights. Here, we show interferometric, free-solution, label-free studies report conformation and hydration changes, and present a new way for interpreting these methods. Intrinsic property changes are the mechanism allowing for unprecedented sensitivities (picomolar to femtomolar) in complex milieu, even when individual binding partners are undetectable. We establish that the existing theory for label-free assay methods such as surface plasmon resonance (SPR) is not applicable and propose a model for the free-solution response function (FreeSRF), validated and highly predictive when combined with quality structural data and reliable calculations of solvent-addressable surface area. The model allows for interpretation of solution-phase, label-free interactions and could facilitate obtaining structural information from a simple mix-and-read assay. (See pp. E1595–E1604.)
Posttranslational regulation of coordinated enzyme activities in the Pup-proteasome system
Yifat Elharar, Ziv Roth, Nir Hecht, Ron Rotkopf, Isam Khalaila, and Eyal Gur
This work describes how a basic regulatory challenge, namely the balancing of two opposing activities of a single enzyme while coordinating these actions with the activity of another enzyme, is addressed in a biological system. In the Pup-proteasome system, the bacterial parallel of the ubiquitin-proteasome system, Pup modification by Dop (deamidase of Pup) is a prerequisite for pupylation by proteasome accessory factor A. Paradoxically, Dop also reverses pupylation through its ability to depupylate substrates. We show that this challenge is resolved posttranslationally by combining substrate discrimination based on molecular weight, and by regulating enzyme levels in response to external stimuli. These findings provide an example of how directionality and coordination of enzyme activities in a single pathway are regulated according to physiological requirements of the cell. (See pp. E1605–E1614.)
Conformational dynamics of a membrane protein chaperone enables spatially regulated substrate capture and release
Fu-Cheng Liang, Gerard Kroon, Camille Z. McAvoy, Chris Chi, Peter E. Wright, and Shu-ou Shan
Molecular chaperones play key roles in maintaining protein homeostasis within cells. Membrane protein chaperones face particular challenges, as they not only protect highly aggregation-prone membrane protein substrates, but also need to achieve tight spatiotemporal coordination of their chaperone cycle. In this work, biochemical and NMR analyses address these questions and for the first time, to our knowledge, define the complete chaperone cycle for cpSRP43, an ATP-independent chaperone dedicated to integral membrane proteins. The study reveals that cpSRP43’s substrate binding domain samples at least three distinct conformations. This property enables it to be readily switched on by positive regulators in the soluble phase to ensure tight substrate binding and be switched off by the translocase at the membrane to ensure facile and productive substrate release. (See pp. E1615–E1624.)
Dual function of C/D box small nucleolar RNAs in rRNA modification and alternative pre-mRNA splicing
Marina Falaleeva, Amadis Pages, Zaneta Matuszek, Sana Hidmi, Lily Agranat-Tamir, Konstantin Korotkov, Yuval Nevo, Eduardo Eyras, Ruth Sperling, and Stefan Stamm
C/D box small nucleolar RNAs (SNORDs) are abundant, short, nucleoli-residing, noncoding RNAs that guide the methyltransferase fibrillarin to perform 2′-O-methylation of target RNAs. We identified 29 SNORDs present in a fibrillarin-containing fraction as well as a fibrillarin-free fraction enriched in spliceosomes. One of these SNORDs, SNORD27, directs rRNA methylation and regulates alternative pre-mRNA splicing (AS) of E2F7 pre-mRNA, a transcriptional repressor of cell cycle-regulated genes. SNORD27 likely regulates E2F7 pre-mRNA AS by masking splice sites through base pairing. This previously unidentified function of SNORDs increases the number of factors regulating AS, a critical step in the expression of the vast majority of human genes, and highlights a potential coupling between AS, cell cycle, proliferation, and ribosome biogenesis. (See pp. E1625–E1634.)
Kinesin-12 motors cooperate to suppress microtubule catastrophes and drive the formation of parallel microtubule bundles
Hauke Drechsler and Andrew D. McAinsh
During cell division, molecular motors from the kinesin superfamily, in particular Kinesin-12 (Kif15) and Kinesin-5 (Eg5), play a crucial role in formation of the spindle—a bipolar microtubule array that is essential for accurate chromosome segregation. While Eg5 is well studied, the mechanism by which Kif15 maintains spindle bipolarity in the absence of Eg5 is unknown. In this work we reconstitute Kif15 motors on dynamic microtubules in vitro. We reveal that Kif15 is a multi-function motor that cross-links microtubules and drives transport of one along the other, which results in the formation of parallel microtubule bundles. Additionally, motors track the microtubule tips, maintaining them in a growing state, which can synchronize microtubule dynamics within a bundle. (See pp. E1635–E1644.)
Actomyosin dynamics drive local membrane component organization in an in vitro active composite layer
Darius Vasco Köster, Kabir Husain, Elda Iljazi, Abrar Bhat, Peter Bieling, R. Dyche Mullins, Madan Rao, and Satyajit Mayor
This manuscript addresses the role of active processes in the spatial organization and dynamics of cell surface components. Using a reconstituted minimal system, we provide experimental evidence for a proposed clustering mechanism that relies on the intrinsic, active mechanics of actin filaments and myosin motors expected to be present at the cell cortex. The coupling between the actomyosin and the lipid bilayer gives rise to an emergent active composite with properties that resemble those observed in live cells. This clustering mechanism is a key feature of the active composite cell surface model and furthers our understanding of the multiple ways in which the cell surface might regulate its composition. (See pp. E1645–E1654.)
Orange carotenoid protein burrows into the phycobilisome to provide photoprotection
Dvir Harris, Ofir Tal, Denis Jallet, Adjélé Wilson, Diana Kirilovsky, and Noam Adir
Protection from overexcitation is one of the most important requirements of all photosynthetic organisms. Here we present a model based on coupled cross-linking/mass spectrometry and site-directed mutagenesis of the means by which the orange carotenoid protein (OCP) binds to the phycobilisome (PBS) antenna complex to avoid photodamage. The model shows that the protein must actively burrow into the complex, separating the PBS rings in the process. This penetration explains for the first time, to our knowledge, how the OCP carotenoid could approach the PBS chromophores at a distance that enables nonphotochemical quenching. However, the alteration in the core structure caused by OCP binding could also prevent energy transmission to the reaction centers. (See pp. E1655–E1662.)
Population-based 3D genome structure analysis reveals driving forces in spatial genome organization
Harianto Tjong, Wenyuan Li, Reza Kalhor, Chao Dai, Shengli Hao, Ke Gong, Yonggang Zhou, Haochen Li, Xianghong Jasmine Zhou, Mark A. Le Gros, Carolyn A. Larabell, Lin Chen, and Frank Alber
We provide a method for population-based structure modeling of whole diploid genomes using Hi-C data. The method considers the stochastic nature of chromosome structures, which allows a detailed analysis of the dynamic landscape of genome organizations. We predict and experimentally validate the presence of chromosome-specific higher-order centromere clusters, which can play a key role in the spatial organization of the human genome, specifically influencing the overall chromosome positioning, as well as the preference of specific chromosome conformations. Our approach generate predictive structural models of diploid genomes from Hi-C data, which can provide insights into the guiding principles of 3D genome organizations. (See pp. E1663–E1672.)
Circadian control of oscillations in mitochondrial rate-limiting enzymes and nutrient utilization by PERIOD proteins
Adi Neufeld-Cohen, Maria S. Robles, Rona Aviram, Gal Manella, Yaarit Adamovich, Benjamin Ladeuix, Dana Nir, Liat Rousso-Noori, Yael Kuperman, Marina Golik, Matthias Mann, and Gad Asher
Mitochondria are major cellular energy suppliers and have to cope with changes in nutrient supply and energy demand that naturally occur throughout the day. We obtained the first, to our knowledge, comprehensive mitochondrial proteome around the clock and identified extensive oscillations in mitochondrial protein abundance that predominantly peak during the early light phase. Remarkably, several rate-limiting mitochondrial enzymes that process different nutrients accumulate in a diurnal manner and are dependent on the clock proteins PER1/2. Concurrently, we uncovered daily oscillations in mitochondrial respiration that are substrate-specific and peak during different times of the day. We propose that the circadian clock PERIOD proteins regulate the diurnal utilization of different nutrients by the mitochondria and thus, optimize mitochondrial function to daily changes in energy supply/demand. (See pp. E1673–E1682.)
Unfolded protein response regulates yeast small GTPase Arl1p activation at late Golgi via phosphorylation of Arf GEF Syt1p
Jia-Wei Hsu, Pei-Hua Tang, I-Hao Wang, Chia-Lun Liu, Wen-Hui Chen, Pei-Chin Tsai, Kuan-Yu Chen, Kuan-Jung Chen, Chia-Jung Yu, and Fang-Jen S. Lee
The unfolded protein response (UPR) is a cellular response to stress caused by accumulation of unfolded or misfolded proteins in the endoplasmic reticulum lumen. ADP ribosylation factor (Arf) GTPases are key regulators of membrane traffic in the Golgi complex. In yeast, Arf guanine nucleotide-exchange factor (GEF) Syt1p activates the Arf-like protein Arl1 at late Golgi, thereby recruiting golgin Imh1p, but how Syt1p GEF activity is regulated is largely unknown. We demonstrate that UPR signaling regulated phosphorylation of Syt1, which is critical for Arl1p activation, recruitment of golgin protein Imh1p to the Golgi, and Syt1p interaction with Arl1. These findings reveal a previously unsuspected relationship between the UPR and Golgi transport. (See pp. E1683–E1690.)
Topologically associated domains enriched for lineage-specific genes reveal expression-dependent nuclear topologies during myogenesis
Daniel S. Neems, Arturo G. Garza-Gongora, Erica D. Smith, and Steven T. Kosak
Genome biology aims to gain insight into nuclear function through the study of genome architecture. Analysis of the completed sequences of various eukaryotic genomes indicates that genes are nonrandomly distributed along chromosomes. Recent molecular approaches based on chromosome confirmation capture have identified topologically associated domains (TADs) as a unifying structural model for chromatin organization; however, whether linear gene order and TADs intersect to affect nuclear organization remains to be resolved. Using human myogenesis as a model, we found that a population of TADs have significant enrichment for myogenic-specific genes that results in changes in their subnuclear and intrachromosomal territory structure. We found that these changes in organization impact biallelic transcription and require cell division to be established. (See pp. E1691–E1700.)
Prediction, dynamics, and visualization of antigenic phenotypes of seasonal influenza viruses
Richard A. Neher, Trevor Bedford, Rodney S. Daniels, Colin A. Russell, and Boris I. Shraiman
Humans mount an antibody-mediated immune response against influenza viruses that can be recalled. Nevertheless, individuals can suffer from recurrent influenza infections as viruses can change their antigenic properties by altering their surface glycoproteins. This antigenic evolution requires frequent update of seasonal influenza vaccines. To inform vaccine updates, laboratories that contribute to the World Health Organization Global Influenza Surveillance and Response System assess the antigenic phenotypes of circulating viruses. Based on the relationship of antigenic distance to genetic differences between viruses, we developed a model to interpret measured antigenic data and predict the properties of viruses that have not been characterized antigenically and explore the model’s value in predicting the future composition of influenza virus populations. (See pp. E1701–E1709.)
ppGpp negatively impacts ribosome assembly affecting growth and antimicrobial tolerance in Gram-positive bacteria
Rebecca M. Corrigan, Lauren E. Bellows, Alison Wood, and Angelika Gründling
When bacteria encounter stresses such as nutrient deprivation, they react by switching on the stringent response, the effects of which are mediated by two nucleotides collectively referred to as (p)ppGpp. These nucleotides function by binding to target proteins, leading to bacterial cells shutting down active growth and entering a state that promotes survival. In Staphylococcus aureus, relatively little is known about the target proteins with which these nucleotides interact. In this work, a genome-wide nucleotide–protein interaction screen was used to identify protein targets of (p)ppGpp to fully establish the pathways these nucleotides control in Gram-positive bacteria. In doing so, we identify several previously unknown targets with roles in ribosomal assembly, cell growth, and antimicrobial tolerance. (See pp. E1710–E1719.)
Focal expression of mutant huntingtin in the songbird basal ganglia disrupts cortico-basal ganglia networks and vocal sequences
Masashi Tanaka (田中雅史), Jonnathan Singh Alvarado, Malavika Murugan, and Richard Mooney
Genetic mutations that impair basal ganglia (BG) function affect sequential movements, but the link between genotype, BG circuit dysfunction, and altered behavior remains unclear. Here, we found that viral expression of a genetic mutation that causes Huntington’s disease (HD) in a vocalization-related region of the songbird BG leads to the selective loss of striatal medium spiny neurons, abnormal temporal patterns of cortico-BG activity during vocalization, and highly unstable vocal sequences. Moreover, silencing activity in the cortical component of this circuit stabilized vocalizations, supporting the idea that the genetic mutation that causes HD affects complex motor sequences by altering temporal patterns of cortico-BG activity. (See pp. E1720–E1727.)
Assessing the sensitivity of diffusion MRI to detect neuronal activity directly
Ruiliang Bai, Craig V. Stewart, Dietmar Plenz, and Peter J. Basser
Diffusion functional MRI has been proposed as a noninvasive neuroimaging method to detect neuronal activity more directly than current blood-oxygen-level-dependent functional MRI, yet initial findings have proven difficult to interpret and reproduce. Here, we study the direct relationship between water diffusion and neuronal activity by simultaneously imaging intracellular calcium using fluorescence along with diffusion MR acquisitions from organotypic rat brain cortex cultures. Although we found that diffusion MR methods can follow the pathological time course of hyperexcitability, e.g., as those seen in epilepsy, they do not appear to be sensitive or specific enough to detect or follow normal neuronal activity. (See pp. E1728–E1737.)
New tools for studying microglia in the mouse and human CNS
Mariko L. Bennett, F. Chris Bennett, Shane A. Liddelow, Bahareh Ajami, Jennifer L. Zamanian, Nathaniel B. Fernhoff, Sara B. Mulinyawe, Christopher J. Bohlen, Aykezar Adil, Andrew Tucker, Irving L. Weissman, Edward F. Chang, Gordon Li, Gerald A. Grant, Melanie G. Hayden Gephart, and Ben A. Barres
Microglia are the tissue resident macrophages of the brain and spinal cord, implicated in important developmental, homeostatic, and disease processes, although our understanding of their roles is complicated by an inability to distinguish microglia from related cell types. Although they share many features with other macrophages, microglia have distinct developmental origins and functions. Here we validate a stable and robustly expressed microglial marker for both mouse and human, transmembrane protein 119 (Tmem119). We use custom-made antibodies against Tmem119 to perform deep RNA sequencing of developing microglia, and demonstrate that microglia mature by the second postnatal week in mice. The antibodies, cell isolation methods, and RNAseq profiles presented here will greatly facilitate our understanding of microglial function in health and disease. (See pp. E1738–E1746.)
Perceptual learning of degraded speech by minimizing prediction error
Ediz Sohoglu and Matthew H. Davis
Experience-dependent changes in sensory processing are critical for successful perception in dynamic and noisy environments. However, the neural and computational mechanisms supporting such changes have remained elusive. Using electrical and magnetic recordings of human brain activity, we demonstrate that two different sources of experience-dependent improvement in perception of degraded speech—the immediate effects of prior knowledge and longer-term changes resulting from perceptual learning—arise from common neural machinery in the sensory cortex although they operate over dramatically different behavioral timescales. Our findings support a specific neuro-computational account of perception in which a single mechanism, minimization of prediction error, drives both the immediate perceptual effects of prior knowledge and longer-term perceptual learning. (See pp. E1747–E1756.)
Discovery and dissection of metabolic oscillations in the microaerobic nitric oxide response network of Escherichia coli
Jonathan L. Robinson and Mark P. Brynildsen
Many bacteria use NO· dioxygenase and NO· reductase to defend themselves against immune-generated NO·. The importance and contribution of these systems under microaerobic conditions, which pathogens are likely to encounter within a host, remain poorly understood. We investigated the NO· response of Escherichia coli throughout the microaerobic regime, and discovered conditions that largely disabled the NO· defenses of E. coli, and environments where the [NO·] oscillated. Components found to comprise the oscillatory circuit are distributed broadly among bacterial species, suggesting that these dynamics could be a characteristic feature of how bacteria respond to NO· in low O2 environments. In support of this hypothesis, analogous oscillations were observed in NO·-stressed cultures of Pseudomonas aeruginosa under low O2 conditions. (See pp. E1757–E1766.)