Noninvasive detection of macrophage activation with single-cell resolution through machine learning
Nicolas Pavillon, Alison J. Hobro, Shizuo Akira, and Nicholas I. Smith
We developed a method enabling the noninvasive study of fine cellular responses that we applied to macrophage activation. The technique is based on a multimodal label-free microscopy system that simultaneously retrieves both morphological and molecular information based on quantitative phase imaging and Raman spectroscopy, respectively. The parameters obtained from these measurements are processed through a machine learning algorithm that makes it possible to reliably assess the macrophage activation state at singlecell level. We found that while each parameter set (morphology and Raman) can detect the activation state, they provide complementary information. Morphology is symptomatic of downstream phenotypes that make the detection dosedependent, while Raman is indicative of upstream molecular changes that enable the detection of selective inhibition of activation pathways. (See pp. E2676–E2685.)
Matching material and cellular timescales maximizes cell spreading on viscoelastic substrates
Ze Gong, Spencer E. Szczesny, Steven R. Caliari, Elisabeth E. Charrier, Ovijit Chaudhuri, Xuan Cao, Yuan Lin, Robert L. Mauck, Paul A. Janmey, Jason A. Burdick, and Vivek B. Shenoy
It is well known that cell proliferation, differentiation, and migration depend strongly on the mechanical stiffness of the extracellular matrix (ECM). Natural ECMs also exhibit dissipative (i.e., plastic, viscoelastic) properties, which can modulate cellular behavior. However, to fully utilize this information in bioengineering applications, a systematic understanding of the role of substrate viscosity on cell function is needed. Using combined theoretical and experimental approaches, we demonstrated that viscous dissipation can be as important as elasticity in determining cell response. Specifically, we found that intermediate viscosity maximizes cell spreading on soft substrates, while cell spreading is independent of viscosity on stiff substrates. This information can now be used to design dissipative biomaterials for optimal control of cell behavior. (See pp. E2686–E2695.)
Structurally modulated codelivery of siRNA and Argonaute 2 for enhanced RNA interference
Jiahe Li, Connie Wu, Wade Wang, Yanpu He, Elad Elkayam, Leemor Joshua-Tor, and Paula T. Hammond
Small interfering RNA (siRNA) has great potential to specifically target undruggable genes in many diseases. Despite its great promise, few siRNAs have been clinically approved due to limited therapeutic efficacy. We propose that one major barrier to RNA interference (RNAi)-based therapy is that the siRNA must form a bound complex with a “scissor” protein, Argonaute 2 (Ago2), after it is released in the cytoplasm. We take the approach of preassembling the duplex siRNA with Ago2 protein, and then packaging the complex with commercial transfection reagents or structurally defined polycations that specifically enhance the clustering of siRNA with Ago2. This unique approach augments gene silencing in cell lines and a melanoma mouse model, providing a potential translatable platform for RNAi therapies. (See pp. E2696–E2705.)
Structural basis for the role of serine-rich repeat proteins from Lactobacillus reuteri in gut microbe–host interactions
Saannya Sequeira, Devon Kavanaugh, Donald A. MacKenzie, Tanja Šuligoj, Samuel Walpole, Charlotte Leclaire, A. Patrick Gunning, Dimitrios Latousakis, William G. T. Willats, Jesus Angulo, Changjiang Dong, and Nathalie Juge
Gut bacteria play a key role in health and disease, but the molecular mechanisms underpinning their interaction with the host remain elusive. The serine-rich repeat proteins (SRRPs) are a family of adhesins identified in many Gram-positive pathogenic bacteria. We previously showed that beneficial bacterial species found in the gut also express SRRPs and that SRRP was required for the ability of Lactobacillus reuteri strain to colonize mice. Here, our structural and biochemical data reveal that L. reuteri SRRP adopts a β-solenoid fold not observed in other structurally characterized SRRPs and functions as an adhesin via a pH-dependent mechanism, providing structural insights into the role of these adhesins in biofilm formation of gut symbionts. (See pp. E2706–E2715.)
N-terminal arginylation generates a bimodal degron that modulates autophagic proteolysis
Young Dong Yoo, Su Ran Mun, Chang Hoon Ji, Ki Woon Sung, Keum Young Kang, Ah Jung Heo, Su Hyun Lee, Jee Young An, Joonsung Hwang, Xiang-Qun Xie, Aaron Ciechanover, Bo Yeon Kim, and Yong Tae Kwon
Conjugation of the amino acid l-arginine (l-Arg) to the protein N termini is a universal posttranslational modification in eukaryotes, yet its functions remain poorly understood. Previous studies showed that the N-terminal Arg of arginylated substrates is bound by N-recognins to induce substrate ubiquitination and proteasomal degradation via the N-end rule pathway of the ubiquitin (Ub)-proteasome system (UPS). Here, we show that the same Nt-Arg residues of arginylated proteins modulate proteolytic flux via macroautophagy when misfolded proteins accumulate beyond the UPS’s capacity. Their Nt-Arg residues bind and allosterically activate the autophagic adaptor p62/STQSM/Sequestosome-1, facilitating cargo collection and lysosomal degradation. Our results suggest that the Nt-Arg proteome of arginylated proteins contributes to reprogramming global proteolytic flux when the UPS is in trouble. (See pp. E2716–E2724.)
Liver X receptor β regulates the development of the dentate gyrus and autistic-like behavior in the mouse
Yulong Cai, Xiaotong Tang, Xi Chen, Xin Li, Ying Wang, Xiaohang Bao, Lian Wang, Dayu Sun, Jinghui Zhao, Yan Xing, Margaret Warner, Haiwei Xu, Jan-Åke Gustafsson, and Xiaotang Fan
Defects in the neurogenesis of the dentate gyrus (DG) seem to be involved in the genesis of autism spectrum disorders (ASD)-like behaviors. Our study reveals that deletion of the Liver X receptor β (LXRβ) in mice causes hypoplasia in the DG, including abnormalities in the formation of progenitor cells and reduced neurogenesis. Behavioral analysis of LXRβ-deficient mice showed autistic-like behaviors, including social interaction deficits and repetitive behavior. These findings provide evidence that early changes in DG neurogenesis is possibly associated with the genesis of autism-related behaviors in LXRβ-deficient mice. (See pp. E2725–E2733.)
dCas9-targeted locus-specific protein isolation method identifies histone gene regulators
Chiahao Tsui, Carla Inouye, Michaella Levy, Andrew Lu, Laurence Florens, Michael P. Washburn, and Robert Tjian
Identifying proteins selectively associated with a genomic locus provides an important entry point toward understanding how a specific gene is regulated. Over the years, there have been several reports describing targeted chromatin-purification methods. However, none has been widely adopted due to the complexity and investment required for such protocols. Here, we present an adaptable chromatin purification system, CLASP, that capitalizes on the versatility of purified dCas9 RNA/protein complexes. We deployed CLASP to purify and identify proteins associated with telomere sequences in human cells as a proof of concept. Next, we targeted a different genomic locus, the Drosophila melanogaster histone cluster, and identified several regulators of the essential histone locus and validated their functional association with genes within the locus. (See pp. E2734–E2741.)
Conformational transitions of the sodium-dependent sugar transporter, vSGLT
Aviv Paz, Derek P. Claxton, Jay Prakash Kumar, Kelli Kazmier, Paola Bisignano, Shruti Sharma, Shannon A. Nolte, Terrin M. Liwag, Vinod Nayak, Ernest M. Wright, Michael Grabe, Hassane S. Mchaourab, and Jeff Abramson
Transporters isomerize between conformations to shuttle cargo across membranes, but the mechanism is not understood. Double electron–electron resonance measurements on the sodium-dependent sugar transporter (vSGLT) were used to explore the conformational state of the transporter under specific ligand conditions. Although sugar transport by vSGLT is driven by sodium gradients, vSGLT adopts an inward-open conformation irrespective of the presence of sodium. In the presence of sodium and galactose, the transporter transitions to an occluded conformation. We propose that the cell’s negative membrane potential aids in driving vSGLT toward the outward-facing state to bind sugar and begin the transport cycle. These findings could be applicable to other transporters whereby the inherent cellular membrane potential is integrated into the transport cycle. (See pp. E2742–E2751.)
BRICHOS domain of Bri2 inhibits islet amyloid polypeptide (IAPP) fibril formation and toxicity in human beta cells
Marie E. Oskarsson, Erik Hermansson, Ye Wang, Nils Welsh, Jenny Presto, Jan Johansson, and Gunilla T. Westermark
Accumulation of islet amyloid polypeptide (IAPP)-containing amyloid fibrils is the main pathological finding in pancreatic islets in type 2 diabetes. The formation of these IAPP amyloid fibrils is considered toxic and may constitute a major cause for the loss of insulin-producing beta cells. The protein domain BRICHOS is present in several different proproteins and possesses antiamyloid chaperone activity. This study demonstrates expression of the BRICHOS-containing protein Bri2 in human pancreatic beta cells and its colocalization with IAPP. The Bri2 BRICHOS domain effectively prevents IAPP from forming fibrils and protects cells from the toxicity associated with IAPP fibrillation. It is concluded that the Bri2 BRICHOS domain may act as an endogenous inhibitor of IAPP amyloid formation in pancreatic beta cells. (See pp. E2752–E2761.)
Transmembrane E3 ligase RNF183 mediates ER stress-induced apoptosis by degrading Bcl-xL
Yanfang Wu, Xia Li, Junying Jia, Yanpeng Zhang, Jing Li, Zhengmao Zhu, Huaqing Wang, Jie Tang, and Junjie Hu
The accumulation of unfolded proteins in the endoplasmic reticulum (ER) is a pathological condition observed in many diseases, including cancer, diabetes, and neurodegenerative diseases. Failure to relieve the cellular stress via adaptive mechanisms of the unfolded protein response (UPR) activates apoptotic cell death. We demonstrate that a membrane-anchored RING finger protein, RNF183, is specifically induced by prolonged ER stress. RNF183 is regulated by IRE1, mainly through miR-7. As an E3 ligase, RNF183 ubiquitinates Bcl-xL, causing its degradation and subsequent apoptosis. Our findings imply that manipulation of RNF183 activity may help control cell fate in pathological conditions and suggest that the close contact between the ER and mitochondria plays a key role in cellular signaling. (See pp. E2762–E2771.)
MAPK signaling couples SCF-mediated degradation of translational regulators to oocyte meiotic progression
Edyta Kisielnicka, Ryuji Minasaki, and Christian R. Eckmann
RNA-binding proteins (RBPs) are key regulators of gene expression. Notably, germ cells deploy many distinct RBPs to guide their differentiation into haploid gametes. While the RNA-regulatory functions of RBPs are emerging, mechanisms controlling their activities and abundance are poorly understood. Due to its highly refined spatial organization, the gonad of Caenorhabditis elegans is an excellent model system to study temporal controls of RBP abundance. We used it to uncover a kinase-mediated protein-turnover axis that is built of evolutionarily conserved components, arranged in a unique manner, to couple RBP-target mRNA de-repression with RBP elimination at pachytene exit, a critical stage in early meiosis. This work reveals how meiotic cell cycle progression is coupled to tissue-specific differentiation events via signaling-induced RBP turnover. (See pp. E2772–E2781.)
Modeling environmentally mediated rotavirus transmission: The role of temperature and hydrologic factors
Alicia N. M. Kraay, Andrew F. Brouwer, Nan Lin, Philip A. Collender, Justin V. Remais, and Joseph N. S. Eisenberg
Although rotavirus persists in water and its incidence varies with temperature, the potential role of waterborne transmission in explaining the observed seasonal pattern in low-income countries in the tropics has thus far been neglected. Our analysis suggests that water can affect incidence for larger communities that draw water from slow-moving or stagnant sources and is likely most important in cooler seasons. Water can also spread outbreaks between communities, even when environmental transmission cannot sustain outbreaks. Our model may help explain temperature–incidence associations found in prior meta-analyses in the tropics, where humidity is high year-round, and shows that the effect of temperature on risk depends on local hydrologic conditions. These findings help explain why the effect of temperature might be context specific. (See pp. E2782–E2790.)
Metabolic shift from glycogen to trehalose promotes lifespan and healthspan in Caenorhabditis elegans
Yonghak Seo, Samuel Kingsley, Griffin Walker, Michelle A. Mondoux, and Heidi A. Tissenbaum
Increased added sugar is contributing to a rise in aging-related diseases. Here, we use the nematode, Caenorhabditis elegans, which store sugar as both glycogen and trehalose. We demonstrate that by modifying sugar storage, we can prevent the harmful effects of a high-sugar diet. Our data show that a metabolic shift increasing the glucose disaccharide trehalose, and decreasing the glucose polysaccharide glycogen, extends lifespan and promotes healthy aging. The positive effects of trehalose require the DAF-16 transcription factor and the process of autophagy. Our data reveal the benefits of trehalose for prolonged health in the face of our high-sugar environment. (See pp. E2791–E2800.)
Myeloid ERK5 deficiency suppresses tumor growth by blocking protumor macrophage polarization via STAT3 inhibition
Emanuele Giurisato, Qiuping Xu, Silvia Lonardi, Brian Telfer, Ilaria Russo, Adam Pearson, Katherine G. Finegan, Wenbin Wang, Jinhua Wang, Nathanael S. Gray, William Vermi, Zhengui Xia, and Cathy Tournier
Macrophages can be functionally reprogrammed by the tumor microenvironment to further tumor growth and malignancy. In this study, we have discovered that this pathological process is dependent on the ERK5 MAPK. Accordingly, we demonstrated that inactivation of ERK5 in macrophages blocked the phosphorylation of STAT3, a transcription factor crucial for determining macrophage polarity, and impaired the growth of melanoma and carcinoma grafts. These results raise the possibility that targeting protumor macrophages via anti-ERK5 therapy constitutes a very attractive strategy for cancer treatment. This is important given that the detection of large numbers of macrophages in human tumors often correlates with poor prognosis, but also with a poor response of the tumor to anticancer agents. (See pp. E2801–E2810.)
Peptides of pHLIP family for targeted intracellular and extracellular delivery of cargo molecules to tumors
Linden C. Wyatt, Anna Moshnikova, Troy Crawford, Donald M. Engelman, Oleg A. Andreev, and Yana K. Reshetnyak
Targeted delivery has been limited by reliance on tumor cell biomarkers. The emergence of the pH (low) insertion peptide (pHLIP) technology provides an alternative by targeting a metabolic marker, tumor cell surface acidity. We report several new pHLIPs, including a new concept, pHLIP bundles, and we evaluate these constructs alongside a new generation of pHLIPs. We also discuss challenges inherent to the design and accurate evaluation of pHLIPs. Our research elucidates the strengths and weaknesses of existing pHLIPs, proposes future peptide modifications that could further improve tumor targeting, and discusses the applicability of this new generation of pHLIPs for specific areas of drug delivery. The principles and new constructs promise to advance applications to tumor therapy. (See pp. E2811–E2818.)
Structure-based discovery of glycomimetic FmlH ligands as inhibitors of bacterial adhesion during urinary tract infection
Vasilios Kalas, Michael E. Hibbing, Amarendar Reddy Maddirala, Ryan Chugani, Jerome S. Pinkner, Laurel K. Mydock-McGrane, Matt S. Conover, James W. Janetka, and Scott J. Hultgren
The emergence of multidrug-resistant bacteria, including uropathogenic Escherichia coli (UPEC), makes the development of targeted antivirulence therapeutics a critical focus of research. During urinary tract infections (UTIs), UPEC uses chaperone–usher pathway pili tipped with an array of adhesins that recognize distinct receptors with sterochemical specificity to facilitate persistence in various tissues and habitats. We used an interdisciplinary approach driven by structural biology and synthetic glycoside chemistry to design and optimize glycomimetic inhibitors of the UPEC adhesin FmlH. These inhibitors competitively blocked FmlH in vitro, in in vivo mouse UTI models, and in ex vivo healthy human kidney tissue. This work demonstrates the utility of structure-driven drug design in the effort to develop antivirulence therapeutic compounds. (See pp. E2819–E2828.)
Communicating the nutritional value of sugar in Drosophila
Farhan Abu, Justin G. Wang, Yangkyun Oh, Jingjing Deng, Thomas A. Neubert, and Greg S. B. Suh
Fruit flies release a previously unreported signal when they consume nutritive food. This signal communicates the presence of nutritive food to other flies and guides them to aggregate around the nutritive food. The nutritional value of food, rather than its taste, is critical for the release of calorie-induced secreted factor (CIF) from the gut end. This signal is not detected by the olfactory system and is not limited to one species in Drosophila. We propose that CIF acts as an ethologically important pheromone that informs nutritional food sources to other flies in the wild. (See pp. E2829–E2838.)
BEST1 gene therapy corrects a diffuse retina-wide microdetachment modulated by light exposure
Karina E. Guziewicz, Artur V. Cideciyan, William A. Beltran, András M. Komáromy, Valerie L. Dufour, Malgorzata Swider, Simone Iwabe, Alexander Sumaroka, Brian T. Kendrick, Gordon Ruthel, Vince A. Chiodo, Elise Héon, William W. Hauswirth, Samuel G. Jacobson, and Gustavo D. Aguirre
One of the most common forms of monogenic macular degeneration worldwide is caused by dominant or recessive bestrophinopathies associated with mutations in the BEST1 gene. Disease expression is known to start with a retina-wide electrophysiological defect leading to localized vitelliform and atrophic lesions and vision loss. To develop lasting therapies for this incurable disease, there is a need for greater understanding of the early pathophysiology before lesion formation. Here we find that the loss of retinal pigment epithelium apical microvilli and resulting microdetachment of the retina represent the earliest features of canine bestrophinopathies. We show that retinal light exposure expands, and dark adaptation contracts, the microdetachments. Subretinal adeno-associated virus-based gene therapy corrects both the vitelliform lesions and the light-modulated microdetachments. (See pp. E2839–E2848.)
Murine knockin model for progranulin-deficient frontotemporal dementia with nonsense-mediated mRNA decay
Andrew D. Nguyen, Thi A. Nguyen, Jiasheng Zhang, Swathi Devireddy, Ping Zhou, Anna M. Karydas, Xialian Xu, Bruce L. Miller, Frank Rigo, Shawn M. Ferguson, Eric J. Huang, Tobias C. Walther, and Robert V. Farese Jr.
Mutations in the GRN gene cause frontotemporal dementia, a devastating neurological disease. The majority of these GRN mutations are nonsense and frameshift mutations. Here, we generated a knockin mouse model with a Grn mutation corresponding to the most prevalent human disease mutation, GRNR493X. We show that mice harboring this mutation phenocopy progranulin-deficient mice, and that the mutation triggers mRNA decay and, as a consequence, low production of Grn. However, the truncated mutant protein that would be produced from this allele is functional, suggesting inhibiting mRNA decay as a therapeutic approach for individuals with progranulin-deficient frontotemporal dementia caused by nonsense mutations. (See pp. E2849–E2858.)
hnRNP R and its main interactor, the noncoding RNA 7SK, coregulate the axonal transcriptome of motoneurons
Michael Briese, Lena Saal-Bauernschubert, Changhe Ji, Mehri Moradi, Hanaa Ghanawi, Michael Uhl, Silke Appenzeller, Rolf Backofen, and Michael Sendtner
Neurons are highly polarized cells. RNA-binding proteins contribute to this polarization by generating diverse subcellular transcriptomes. The RNA-binding protein hnRNP R is essential for axon growth in motoneurons. This study reports the RNA interactome for hnRNP R. The main interacting RNA of hnRNP R was the noncoding RNA 7SK. Depletion of 7SK from primary motoneurons disturbed axon growth. This effect was dependent on the interaction of 7SK with hnRNP R. Both hnRNP R and 7SK localize to axons. Loss of 7SK led to a similar depletion of axonal transcripts as loss of hnRNP R. Our data suggest that 7SK, in addition to its role in transcriptional regulation, acts in concert with hnRNP R to sort specific transcripts into axons. (See pp. E2859–E2868.)
Simultaneous imaging and functional studies reveal a tight correlation between calcium and actin networks
Carlisle S. Bascom Jr., Lawrence J. Winship, and Magdalena Bezanilla
Some plant cells, such as root hairs, pollen tubes, and moss protonemata, expand in a highly polarized manner known as tip growth. That calcium and actin play essential roles in tip growth has been well established, but how they interact with one another in vivo has mostly been modeled from in vitro studies. Here, we use a wavelet analysis approach to dissect the complex calcium homeostasis found in the moss Physcomitrella patens. By perturbing the actin cytoskeleton with drugs and targeted genome editing we find that the shortest calcium oscillation is directly coupled to actin dynamics. This approach to analyzing calcium oscillation profiles sets the stage for subtle dissection of the calcium–actin interaction mechanism. (See pp. E2869–E2878.)
Integration of speed and time for estimating time to contact
Chia-Jung Chang and Mehrdad Jazayeri
Existing theories suggest that reacting to dynamic stimuli is made possible by relying on internal estimates of kinematic variables. For example, to catch a bouncing ball the brain relies on the position and speed of the ball. However, when kinematic information is unreliable one may additionally rely on temporal cues. In the bouncing ball example, when visibility is low one may benefit from the temporal information provided by the sound of the bounces. Our work provides evidence that humans rely on such temporal cues and automatically integrate them with kinematic information to optimize their performance. This finding reveals a hitherto unappreciated role of the brain’s timing mechanisms in sensorimotor function. (See pp. E2879–E2887.)
Nongenetic origins of cell-to-cell variability in B lymphocyte proliferation
Simon Mitchell, Koushik Roy, Thomas A. Zangle, and Alexander Hoffmann
This study addresses why splenic B lymphocytes show differential cell proliferation and death decisions, and whether these may be predictable. Biology provides examples of both stochastic decision making and highly deterministic developmental programs. Prior studies of B lymphocytes suggested these cells make stochastic decisions, but the key experiment—long-term tracing of individual cell lineages—has not been done. Overcoming the technical challenges, we found that B cell fate decisions are largely nonstochastic. Using a mathematical model, we found that they are predictable, as long as the state of the molecular network in founder cells is known. That allowed us to identify the molecular determinants of proliferative fate decisions, which potentially constitute novel drug targets and biomarkers for B cell-mediated diseases. (See pp. E2888–E2897.)