PLOS BiologyPLOShttps://journals.plos.org/plosbiology/webmaster@plos.orgA Peer-Reviewed Open-Access Journalhttps://journals.plos.org/plosbiology/feed/atomAll PLOS articles are Open Access.https://journals.plos.org/plosbiology/resource/img/favicon.icohttps://journals.plos.org/plosbiology/resource/img/favicon.ico2024-03-19T02:21:31ZOn the impact of incomplete taxon sampling on the relative timing of gene transfer eventsMoisès BernabeuSaioa Manzano-MoralesToni Gabaldón10.1371/journal.pbio.30024602024-03-18T14:00:00Z2024-03-18T14:00:00Z<p>by Moisès Bernabeu, Saioa Manzano-Morales, Toni Gabaldón</p>
A recent study questioned the use of branch length methods to assess the relative timing of horizontal gene transfers because of the effects of so-called “ghost” lineages. This Formal Comment discusses key considerations regarding the potential effect of missing lineages when assessing relative timing of evolutionary events.The evolutionary safety of mutagenic drugs should be assessed before drug approvalGabriela LobinskaVyacheslav TretyachenkoOrna DahanMartin A. NowakYitzhak Pilpel10.1371/journal.pbio.30025702024-03-15T14:00:00Z2024-03-15T14:00:00Z<p>by Gabriela Lobinska, Vyacheslav Tretyachenko, Orna Dahan, Martin A. Nowak, Yitzhak Pilpel</p>
Some drugs increase the mutation rate of their target pathogen, a potentially concerning mechanism as the pathogen might evolve faster toward an undesired phenotype. We suggest a four-step assessment of evolutionary safety for the approval of such treatments.
Some drugs increase the mutation rate of their target pathogen, raising the concern that they might thereby increase the pathogen’s rate of adaptation. This Perspective article proposes a four-step process to evaluate the evolutionary safety of new treatments, calling on regulatory authorities to take this into account before approval for widespread use.Developing inhibitory peptides against SARS-CoV-2 envelope proteinRamsey BekdashKazushige YoshidaManoj S. NairLauren QiuJohnathan AhdoutHsiang-Yi TsaiKunihiro UryuRajesh K. SoniYaoxing HuangDavid D. HoMasayuki Yazawa10.1371/journal.pbio.30025222024-03-14T14:00:00Z2024-03-14T14:00:00Z<p>by Ramsey Bekdash, Kazushige Yoshida, Manoj S. Nair, Lauren Qiu, Johnathan Ahdout, Hsiang-Yi Tsai, Kunihiro Uryu, Rajesh K. Soni, Yaoxing Huang, David D. Ho, Masayuki Yazawa</p>
Severe Acute Respiratory Syndrome Coronavirus 2 (SARS-CoV-2) has affected approximately 800 million people since the start of the Coronavirus Disease 2019 (COVID-19) pandemic. Because of the high rate of mutagenesis in SARS-CoV-2, it is difficult to develop a sustainable approach for prevention and treatment. The Envelope (E) protein is highly conserved among human coronaviruses. Previous studies reported that SARS-CoV-1 E deficiency reduced viral propagation, suggesting that E inhibition might be an effective therapeutic strategy for SARS-CoV-2. Here, we report inhibitory peptides against SARS-CoV-2 E protein named iPep-SARS2-E. Leveraging E-induced alterations in proton homeostasis and NFAT/AP-1 pathway in mammalian cells, we developed screening platforms to design and optimize the peptides that bind and inhibit E protein. Using Vero-E6 cells, human-induced pluripotent stem cell-derived branching lung organoid and mouse models with SARS-CoV-2, we found that iPep-SARS2-E significantly inhibits virus egress and reduces viral cytotoxicity and propagation in vitro and in vivo. Furthermore, the peptide can be customizable for E protein of other human coronaviruses such as Middle East Respiratory Syndrome Coronavirus (MERS-CoV). The results indicate that E protein can be a potential therapeutic target for human coronaviruses.Engineering of Cas12a nuclease variants with enhanced genome-editing specificityPeng ChenJin ZhouHuan LiuErchi ZhouBoxiao HeYankang WuHongjian WangZaiqiao SunChonil PaekJun LeiYongshun ChenXinghua ZhangLei Yin10.1371/journal.pbio.30025142024-03-14T14:00:00Z2024-03-14T14:00:00Z<p>by Peng Chen, Jin Zhou, Huan Liu, Erchi Zhou, Boxiao He, Yankang Wu, Hongjian Wang, Zaiqiao Sun, Chonil Paek, Jun Lei, Yongshun Chen, Xinghua Zhang, Lei Yin</p>
The clustered regularly interspaced short palindromic repeat (CRISPR)-Cas12a system is a powerful tool in gene editing; however, crRNA-DNA mismatches might induce unwanted cleavage events, especially at the distal end of the PAM. To minimize this limitation, we engineered a hyper fidelity AsCas12a variant carrying the mutations S186A/R301A/T315A/Q1014A/K414A (termed HyperFi-As) by modifying amino acid residues interacting with the target DNA and crRNA strand. HyperFi-As retains on-target activities comparable to wild-type AsCas12a (AsCas12aWT) in human cells. We demonstrated that HyperFi-As has dramatically reduced off-target effects in human cells, and HyperFi-As possessed notably a lower tolerance to mismatch at the position of the PAM-distal region compared with the wild type. Further, a modified single-molecule DNA unzipping assay at proper constant force was applied to evaluate the stability and transient stages of the CRISPR/Cas ribonucleoprotein (RNP) complex. Multiple states were sensitively detected during the disassembly of the DNA-Cas12a-crRNA complexes. On off-target DNA substrates, the HyperFi-As-crRNA was harder to maintain the R-loop complex state compared to the AsCas12aWT, which could explain exactly why the HyperFi-As has low off-targeting effects in human cells. Our findings provide a novel version of AsCas12a variant with low off-target effects, especially capable of dealing with the high off-targeting in the distal region from the PAM. An insight into how the AsCas12a variant behaves at off-target sites was also revealed at the single-molecule level and the unzipping assay to evaluate multiple states of CRISPR/Cas RNP complexes might be greatly helpful for a deep understanding of how CRISPR/Cas behaves and how to engineer it in future.Polyphosphate kinase regulates LPS structure and polymyxin resistance during starvation in <i>E</i>. <i>coli</i>Kanchi BaijalIryna AbramchukCarmen M. HerreraThien-Fah MahM. Stephen TrentMathieu Lavallée-AdamMichael Downey10.1371/journal.pbio.30025582024-03-13T14:00:00Z2024-03-13T14:00:00Z<p>by Kanchi Baijal, Iryna Abramchuk, Carmen M. Herrera, Thien-Fah Mah, M. Stephen Trent, Mathieu Lavallée-Adam, Michael Downey</p>
Polyphosphates (polyP) are chains of inorganic phosphates that can reach over 1,000 residues in length. In <i>Escherichia coli</i>, polyP is produced by the polyP kinase (PPK) and is thought to play a protective role during the response to cellular stress. However, the molecular pathways impacted by PPK activity and polyP accumulation remain poorly characterized. In this work, we used label-free mass spectrometry to study the response of bacteria that cannot produce polyP (Δ<i>ppk</i>) during starvation to identify novel pathways regulated by PPK. In response to starvation, we found 92 proteins significantly differentially expressed between wild-type and Δ<i>ppk</i> mutant cells. Wild-type cells were enriched for proteins related to amino acid biosynthesis and transport, while Δ<i>ppk</i> mutants were enriched for proteins related to translation and ribosome biogenesis, suggesting that without PPK, cells remain inappropriately primed for growth even in the absence of the required building blocks. From our data set, we were particularly interested in Arn and EptA proteins, which were down-regulated in Δ<i>ppk</i> mutants compared to wild-type controls, because they play a role in lipid A modifications linked to polymyxin resistance. Using western blotting, we confirm differential expression of these and related proteins in K-12 strains and a uropathogenic isolate, and provide evidence that this mis-regulation in Δ<i>ppk</i> cells stems from a failure to induce the BasRS two-component system during starvation. We also show that Δ<i>ppk</i> mutants unable to up-regulate Arn and EptA expression lack the respective L-Ara4N and pEtN modifications on lipid A. In line with this observation, loss of <i>ppk</i> restores polymyxin sensitivity in resistant strains carrying a constitutively active <i>basR</i> allele. Overall, we show a new role for PPK in lipid A modification during starvation and provide a rationale for targeting PPK to sensitize bacteria towards polymyxin treatment. We further anticipate that our proteomics work will provide an important resource for researchers interested in the diverse pathways impacted by PPK.Specification of distinct cell types in a sensory-adhesive organ important for metamorphosis in tunicate larvaeChristopher J. JohnsonFlorian Razy-KrajkaFan ZengKatarzyna M. PiekarzShweta BiliyaUte RothbächerAlberto Stolfi10.1371/journal.pbio.30025552024-03-13T14:00:00Z2024-03-13T14:00:00Z<p>by Christopher J. Johnson, Florian Razy-Krajka, Fan Zeng, Katarzyna M. Piekarz, Shweta Biliya, Ute Rothbächer, Alberto Stolfi</p>
The papillae of tunicate larvae contribute sensory, adhesive, and metamorphosis-regulating functions that are crucial for the biphasic lifestyle of these marine, non-vertebrate chordates. We have identified additional molecular markers for at least 5 distinct cell types in the papillae of the model tunicate <i>Ciona</i>, allowing us to further study the development of these organs. Using tissue-specific CRISPR/Cas9-mediated mutagenesis and other molecular perturbations, we reveal the roles of key transcription factors and signaling pathways that are important for patterning the papilla territory into a highly organized array of different cell types and shapes. We further test the contributions of different transcription factors and cell types to the production of the adhesive glue that allows for larval attachment during settlement, and to the processes of tail retraction and body rotation during metamorphosis. With this study, we continue working towards connecting gene regulation to cellular functions that control the developmental transition between the motile larva and sessile adult of <i>Ciona</i>.Neuronal ageing is promoted by the decay of the microtubule cytoskeletonPilar Okenve-RamosRory GoslingMonika Chojnowska-MongaKriti GuptaSamuel ShieldsHaifa AlhadyianCeryce CollieEmilia GregoryNatalia Sanchez-Soriano10.1371/journal.pbio.30025042024-03-13T14:00:00Z2024-03-13T14:00:00Z<p>by Pilar Okenve-Ramos, Rory Gosling, Monika Chojnowska-Monga, Kriti Gupta, Samuel Shields, Haifa Alhadyian, Ceryce Collie, Emilia Gregory, Natalia Sanchez-Soriano</p>
Natural ageing is accompanied by a decline in motor, sensory, and cognitive functions, all impacting quality of life. Ageing is also the predominant risk factor for many neurodegenerative diseases, including Parkinson’s disease and Alzheimer’s disease. We need to therefore gain a better understanding of the cellular and physiological processes underlying age-related neuronal decay. However, gaining this understanding is a slow process due to the large amount of time required to age mammalian or vertebrate animal models. Here, we introduce a new cellular model within the <i>Drosophila</i> brain, in which we report classical ageing hallmarks previously observed in the primate brain. These hallmarks include axonal swellings, cytoskeletal decay, a reduction in axonal calibre, and morphological changes arising at synaptic terminals. In the fly brain, these changes begin to occur within a few weeks, ideal to study the underlying mechanisms of ageing. We discovered that the decay of the neuronal microtubule (MT) cytoskeleton precedes the onset of other ageing hallmarks. We showed that the MT-binding factors Tau, EB1, and Shot/MACF1, are necessary for MT maintenance in axons and synapses, and that their functional loss during ageing triggers MT bundle decay, followed by a decline in axons and synaptic terminals. Furthermore, genetic manipulations that improve MT networks slowed down the onset of neuronal ageing hallmarks and confer aged specimens the ability to outperform age-matched controls. Our work suggests that MT networks are a key lesion site in ageing neurons and therefore the MT cytoskeleton offers a promising target to improve neuronal decay in advanced age.Low-cost, versatile, and highly reproducible microfabrication pipeline to generate 3D-printed customised cell culture devices with complex designsCathleen HagemannMatthew C. D. BaileyEugenia CarraroKsenia S. StankevichValentina Maria LionelloNoreen KhokharPacharaporn SuklaiCarmen Moreno-GonzalezKelly O’TooleGeorge KonstantinouChristina L. DixSudeep JoshiEleonora GiagnorioMads S. BergholtChristopher D. SpicerAlbane ImbertFrancesco Saverio TedescoAndrea Serio10.1371/journal.pbio.30025032024-03-13T14:00:00Z2024-03-13T14:00:00Z<p>by Cathleen Hagemann, Matthew C. D. Bailey, Eugenia Carraro, Ksenia S. Stankevich, Valentina Maria Lionello, Noreen Khokhar, Pacharaporn Suklai, Carmen Moreno-Gonzalez, Kelly O’Toole, George Konstantinou, Christina L. Dix, Sudeep Joshi, Eleonora Giagnorio, Mads S. Bergholt, Christopher D. Spicer, Albane Imbert, Francesco Saverio Tedesco, Andrea Serio</p>
Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models while retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices, and microfluidic systems to larger constructs such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques, equipment and tools required for their manufacture, and the expertise necessary, biological and biomedical labs tend more often to rely on already made devices. Indeed, commercially developed devices are available for a variety of applications but are often costly and, importantly, lack the potential for customisation by each individual lab. The last point is quite crucial, as often experiments in wet labs are adapted to whichever design is already available rather than designing and fabricating custom systems that perfectly fit the biological question. This combination of factors still restricts widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent advances in bioengineering and microfabrication aimed at solving these issues, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised a low-cost and highly reproducible microfabrication pipeline. This is thought specifically for biomedical and biological wet labs with not prior experience in the field, which will enable them to generate a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with limited expertise in those techniques and enables the manufacture of complex devices across the μm to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D-printed constructs for PDMS curing, using a combination of polymerisation steps, washes, and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilisation of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multipart hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.Noncanonical and reversible cysteine ubiquitination prevents the overubiquitination of PEX5 at the peroxisomal membraneTânia FranciscoAna G. PedrosaTony A. RodriguesTarad AbalkhailHongli LiMaria J. FerreiraGerbrand J. van der Heden van NoortMarc FransenEwald H. HettemaJorge E. Azevedo10.1371/journal.pbio.30025672024-03-12T14:00:00Z2024-03-12T14:00:00Z<p>by Tânia Francisco, Ana G. Pedrosa, Tony A. Rodrigues, Tarad Abalkhail, Hongli Li, Maria J. Ferreira, Gerbrand J. van der Heden van Noort, Marc Fransen, Ewald H. Hettema, Jorge E. Azevedo</p>
PEX5, the peroxisomal protein shuttling receptor, binds newly synthesized proteins in the cytosol and transports them to the organelle. During its stay at the peroxisomal protein translocon, PEX5 is monoubiquitinated at its cysteine 11 residue, a mandatory modification for its subsequent ATP-dependent extraction back into the cytosol. The reason why a cysteine and not a lysine residue is the ubiquitin acceptor is unknown. Using an established rat liver-based cell-free in vitro system, we found that, in contrast to wild-type PEX5, a PEX5 protein possessing a lysine at position 11 is polyubiquitinated at the peroxisomal membrane, a modification that negatively interferes with the extraction process. Wild-type PEX5 cannot retain a polyubiquitin chain because ubiquitination at cysteine 11 is a reversible reaction, with the E2-mediated deubiquitination step presenting faster kinetics than PEX5 polyubiquitination. We propose that the reversible nonconventional ubiquitination of PEX5 ensures that neither the peroxisomal protein translocon becomes obstructed with polyubiquitinated PEX5 nor is PEX5 targeted for proteasomal degradation.Synaptic and dendritic architecture of different types of hippocampal somatostatin interneuronsVirág TakácsZsuzsanna BardócziÁron OroszAbel MajorLuca TarPéter BerkiPéter PappMárton I. MayerHunor SebőkLuca ZsoltKatalin E. SosSzabolcs KáliTamás F. FreundGábor Nyiri10.1371/journal.pbio.30025392024-03-12T14:00:00Z2024-03-12T14:00:00Z<p>by Virág Takács, Zsuzsanna Bardóczi, Áron Orosz, Abel Major, Luca Tar, Péter Berki, Péter Papp, Márton I. Mayer, Hunor Sebők, Luca Zsolt, Katalin E. Sos, Szabolcs Káli, Tamás F. Freund, Gábor Nyiri</p>
GABAergic inhibitory neurons fundamentally shape the activity and plasticity of cortical circuits. A major subset of these neurons contains somatostatin (SOM); these cells play crucial roles in neuroplasticity, learning, and memory in many brain areas including the hippocampus, and are implicated in several neuropsychiatric diseases and neurodegenerative disorders. Two main types of SOM-containing cells in area CA1 of the hippocampus are oriens-lacunosum-moleculare (OLM) cells and hippocampo-septal (HS) cells. These cell types show many similarities in their soma-dendritic architecture, but they have different axonal targets, display different activity patterns in vivo, and are thought to have distinct network functions. However, a complete understanding of the functional roles of these interneurons requires a precise description of their intrinsic computational properties and their synaptic interactions. In the current study we generated, analyzed, and make available several key data sets that enable a quantitative comparison of various anatomical and physiological properties of OLM and HS cells in mouse. The data set includes detailed scanning electron microscopy (SEM)-based 3D reconstructions of OLM and HS cells along with their excitatory and inhibitory synaptic inputs. Combining this core data set with other anatomical data, patch-clamp electrophysiology, and compartmental modeling, we examined the precise morphological structure, inputs, outputs, and basic physiological properties of these cells. Our results highlight key differences between OLM and HS cells, particularly regarding the density and distribution of their synaptic inputs and mitochondria. For example, we estimated that an OLM cell receives about 8,400, whereas an HS cell about 15,600 synaptic inputs, about 16% of which are GABAergic. Our data and models provide insight into the possible basis of the different functionality of OLM and HS cell types and supply essential information for more detailed functional models of these neurons and the hippocampal network.Recommendations for measuring and standardizing light for laboratory mammals to improve welfare and reproducibility in animal researchRobert J. LucasAnnette E. AllenGeorge C. BrainardTimothy M. BrownRobert T. DauchyAltug DidikogluMichael Tri H. DoBrianna N. GaskillSamer HattarPenny HawkinsRoelof A. HutRichard J. McDowellRandy J. NelsonJan-Bas PrinsTiffany M. SchmidtJoseph S. TakahashiVandana VermaVootele VoikarSara WellsStuart N. Peirson10.1371/journal.pbio.30025352024-03-12T14:00:00Z2024-03-12T14:00:00Z<p>by Robert J. Lucas, Annette E. Allen, George C. Brainard, Timothy M. Brown, Robert T. Dauchy, Altug Didikoglu, Michael Tri H. Do, Brianna N. Gaskill, Samer Hattar, Penny Hawkins, Roelof A. Hut, Richard J. McDowell, Randy J. Nelson, Jan-Bas Prins, Tiffany M. Schmidt, Joseph S. Takahashi, Vandana Verma, Vootele Voikar, Sara Wells, Stuart N. Peirson</p>
Light enables vision and exerts widespread effects on physiology and behavior, including regulating circadian rhythms, sleep, hormone synthesis, affective state, and cognitive processes. Appropriate lighting in animal facilities may support welfare and ensure that animals enter experiments in an appropriate physiological and behavioral state. Furthermore, proper consideration of light during experimentation is important both when it is explicitly employed as an independent variable and as a general feature of the environment. This Consensus View discusses metrics to use for the quantification of light appropriate for nonhuman mammals and their application to improve animal welfare and the quality of animal research. It provides methods for measuring these metrics, practical guidance for their implementation in husbandry and experimentation, and quantitative guidance on appropriate light exposure for laboratory mammals. The guidance provided has the potential to improve data quality and contribute to reduction and refinement, helping to ensure more ethical animal use.IntAct: A nondisruptive internal tagging strategy to study the organization and function of actin isoformsMaxime C. van ZwamAnubhav DharWillem BosmanWendy van StraatenSuzanne WeijersEmiel SetaBen JoostenJeffrey van HarenSaravanan PalaniKoen van den Dries10.1371/journal.pbio.30025512024-03-11T14:00:00Z2024-03-11T14:00:00Z<p>by Maxime C. van Zwam, Anubhav Dhar, Willem Bosman, Wendy van Straaten, Suzanne Weijers, Emiel Seta, Ben Joosten, Jeffrey van Haren, Saravanan Palani, Koen van den Dries</p>
Mammals have 6 highly conserved actin isoforms with nonredundant biological functions. The molecular basis of isoform specificity, however, remains elusive due to a lack of tools. Here, we describe the development of IntAct, an internal tagging strategy to study actin isoforms in fixed and living cells. We identified a residue pair in β-actin that permits tag integration and used knock-in cell lines to demonstrate that IntAct β-actin expression and filament incorporation is indistinguishable from wild type. Furthermore, IntAct β-actin remains associated with common actin-binding proteins (ABPs) and can be targeted in living cells. We demonstrate the usability of IntAct for actin isoform investigations by showing that actin isoform-specific distribution is maintained in human cells. Lastly, we observed a variant-dependent incorporation of tagged actin variants into yeast actin patches, cables, and cytokinetic rings demonstrating cross species applicability. Together, our data indicate that IntAct is a versatile tool to study actin isoform localization, dynamics, and molecular interactions.An ancient bacterial zinc acquisition system identified from a cyanobacterial exoproteomeCristina Sarasa-BuisanJesús A. G. Ochoa de AldaCristina Velázquez-SuárezMiguel Ángel RubioGuadalupe Gómez-BaenaMaría F. FillatIgnacio Luque10.1371/journal.pbio.30025462024-03-11T14:00:00Z2024-03-11T14:00:00Z<p>by Cristina Sarasa-Buisan, Jesús A. G. Ochoa de Alda, Cristina Velázquez-Suárez, Miguel Ángel Rubio, Guadalupe Gómez-Baena, María F. Fillat, Ignacio Luque</p>
Bacteria have developed fine-tuned responses to cope with potential zinc limitation. The Zur protein is a key player in coordinating this response in most species. Comparative proteomics conducted on the cyanobacterium <i>Anabaena</i> highlighted the more abundant proteins in a <i>zur</i> mutant compared to the wild type. Experimental evidence showed that the exoprotein ZepA mediates zinc uptake. Genomic context of the <i>zepA</i> gene and protein structure prediction provided additional insights on the regulation and putative function of ZepA homologs. Phylogenetic analysis suggests that ZepA represents a primordial system for zinc acquisition that has been conserved for billions of years in a handful of species from distant bacterial lineages. Furthermore, these results show that Zur may have been one of the first regulators of the FUR family to evolve, consistent with the scarcity of zinc in the ecosystems of the Archean eon.Proteasome inhibition triggers tissue-specific immune responses against different pathogens in <i>C</i>. <i>elegans</i>Manish GroverSpencer S. GangEmily R. TroemelMichalis Barkoulas10.1371/journal.pbio.30025432024-03-11T14:00:00Z2024-03-11T14:00:00Z<p>by Manish Grover, Spencer S. Gang, Emily R. Troemel, Michalis Barkoulas</p>
Protein quality control pathways play important roles in resistance against pathogen infection. For example, the conserved transcription factor SKN-1/NRF up-regulates proteostasis capacity after blockade of the proteasome and also promotes resistance against bacterial infection in the nematode <i>Caenorhabditis elegans</i>. SKN-1/NRF has 3 isoforms, and the SKN-1A/NRF1 isoform, in particular, regulates proteasomal gene expression upon proteasome dysfunction as part of a conserved bounce-back response. We report here that, in contrast to the previously reported role of SKN-1 in promoting resistance against bacterial infection, loss-of-function mutants in <i>skn-1a</i> and its activating enzymes <i>ddi-1</i> and <i>png-1</i> show constitutive expression of immune response programs against natural eukaryotic pathogens of <i>C</i>. <i>elegans</i>. These programs are the oomycete recognition response (ORR), which promotes resistance against oomycetes that infect through the epidermis, and the intracellular pathogen response (IPR), which promotes resistance against intestine-infecting microsporidia. Consequently, <i>skn-1a</i> mutants show increased resistance to both oomycete and microsporidia infections. We also report that almost all ORR/IPR genes induced in common between these programs are regulated by the proteasome and interestingly, specific ORR/IPR genes can be induced in distinct tissues depending on the exact trigger. Furthermore, we show that increasing proteasome function significantly reduces oomycete-mediated induction of multiple ORR markers. Altogether, our findings demonstrate that proteasome regulation keeps innate immune responses in check in a tissue-specific manner against natural eukaryotic pathogens of the <i>C</i>. <i>elegans</i> epidermis and intestine.Widespread prevalence of a methylation-dependent switch to activate an essential DNA damage response in bacteriaAditya KamatNgat T. TranMohak ShardaNeha SontakkeTung B. K. LeAnjana Badrinarayanan10.1371/journal.pbio.30025402024-03-11T14:00:00Z2024-03-11T14:00:00Z<p>by Aditya Kamat, Ngat T. Tran, Mohak Sharda, Neha Sontakke, Tung B. K. Le, Anjana Badrinarayanan</p>
DNA methylation plays central roles in diverse cellular processes, ranging from error-correction during replication to regulation of bacterial defense mechanisms. Nevertheless, certain aberrant methylation modifications can have lethal consequences. The mechanisms by which bacteria detect and respond to such damage remain incompletely understood. Here, we discover a highly conserved but previously uncharacterized transcription factor (Cada2), which orchestrates a methylation-dependent adaptive response in <i>Caulobacter</i>. This response operates independently of the SOS response, governs the expression of genes crucial for direct repair, and is essential for surviving methylation-induced damage. Our molecular investigation of Cada2 reveals a cysteine methylation-dependent posttranslational modification (PTM) and mode of action distinct from its <i>Escherichia coli</i> counterpart, a trait conserved across all bacteria harboring a Cada2-like homolog instead. Extending across the bacterial kingdom, our findings support the notion of divergence and coevolution of adaptive response transcription factors and their corresponding sequence-specific DNA motifs. Despite this diversity, the ubiquitous prevalence of adaptive response regulators underscores the significance of a transcriptional switch, mediated by methylation PTM, in driving a specific and essential bacterial DNA damage response.Attention to audiovisual speech shapes neural processing through feedback-feedforward loops between different nodes of the speech networkPatrik WikmanViljami SalmelaEetu SjöblomMiika LeminenMatti LaineKimmo Alho10.1371/journal.pbio.30025342024-03-11T14:00:00Z2024-03-11T14:00:00Z<p>by Patrik Wikman, Viljami Salmela, Eetu Sjöblom, Miika Leminen, Matti Laine, Kimmo Alho</p>
Selective attention-related top-down modulation plays a significant role in separating relevant speech from irrelevant background speech when vocal attributes separating concurrent speakers are small and continuously evolving. Electrophysiological studies have shown that such top-down modulation enhances neural tracking of attended speech. Yet, the specific cortical regions involved remain unclear due to the limited spatial resolution of most electrophysiological techniques. To overcome such limitations, we collected both electroencephalography (EEG) (high temporal resolution) and functional magnetic resonance imaging (fMRI) (high spatial resolution), while human participants selectively attended to speakers in audiovisual scenes containing overlapping cocktail party speech. To utilise the advantages of the respective techniques, we analysed neural tracking of speech using the EEG data and performed representational dissimilarity-based EEG-fMRI fusion. We observed that attention enhanced neural tracking and modulated EEG correlates throughout the latencies studied. Further, attention-related enhancement of neural tracking fluctuated in predictable temporal profiles. We discuss how such temporal dynamics could arise from a combination of interactions between attention and prediction as well as plastic properties of the auditory cortex. EEG-fMRI fusion revealed attention-related iterative feedforward-feedback loops between hierarchically organised nodes of the ventral auditory object related processing stream. Our findings support models where attention facilitates dynamic neural changes in the auditory cortex, ultimately aiding discrimination of relevant sounds from irrelevant ones while conserving neural resources.Degrade to stay healthy—Proteolytic interplay during inflammationChristian Münz10.1371/journal.pbio.30025482024-03-07T14:00:00Z2024-03-07T14:00:00Z<p>by Christian Münz</p>
Proteasomes and autophagy constitute the 2 main proteolytic machineries for cytoplasmic content. A new study in PLOS Biology now demonstrates that autophagy stimulation alters proteasome composition, degrading hyperactive immunoproteasomes and thereby limiting inflammation.
Proteasomes and autophagy constitute the two main proteolytic machineries for cytoplasmic content. This Primer explores a new study in PLOS Biology which demonstrates that autophagy stimulation alters proteasome composition, degrading hyperactive immunoproteasomes and thereby limiting inflammation.Role for gene conversion in the evolution of cell-surface antigens of the malaria parasite <i>Plasmodium falciparum</i>Brice LetcherSorina MaciucaZamin Iqbal10.1371/journal.pbio.30025072024-03-07T14:00:00Z2024-03-07T14:00:00Z<p>by Brice Letcher, Sorina Maciuca, Zamin Iqbal</p>
While the malaria parasite <i>Plasmodium falciparum</i> has low average genome-wide diversity levels, likely due to its recent introduction from a gorilla-infecting ancestor (approximately 10,000 to 50,000 years ago), some genes display extremely high diversity levels. In particular, certain proteins expressed on the surface of human red blood cell–infecting merozoites (merozoite surface proteins (MSPs)) possess exactly 2 deeply diverged lineages that have seemingly not recombined. While of considerable interest, the evolutionary origin of this phenomenon remains unknown. In this study, we analysed the genetic diversity of 2 of the most variable MSPs, DBLMSP and DBLMSP2, which are paralogs (descended from an ancestral duplication). Despite thousands of available Illumina WGS datasets from malaria-endemic countries, diversity in these genes has been hard to characterise as reads containing highly diverged alleles completely fail to align to the reference genome. To solve this, we developed a pipeline leveraging genome graphs, enabling us to genotype them at high accuracy and completeness. Using our newly- resolved sequences, we found that both genes exhibit 2 deeply diverged lineages in a specific protein domain (DBL) and that one of the 2 lineages is shared across the genes. We identified clear evidence of nonallelic gene conversion between the 2 genes as the likely mechanism behind sharing, leading us to propose that gene conversion between diverged paralogs, and not recombination suppression, can generate this surprising genealogy; a model that is furthermore consistent with high diversity levels in these 2 genes despite the strong historical <i>P</i>. <i>falciparum</i> transmission bottleneck.FAM81A is a postsynaptic protein that regulates the condensation of postsynaptic proteins via liquid–liquid phase separationTakeshi KaizukaTaisei HirouchiTakeo SaneyoshiToshihiko ShirafujiMark O. CollinsSeth G. N. GrantYasunori HayashiToru Takumi10.1371/journal.pbio.30020062024-03-07T14:00:00Z2024-03-07T14:00:00Z<p>by Takeshi Kaizuka, Taisei Hirouchi, Takeo Saneyoshi, Toshihiko Shirafuji, Mark O. Collins, Seth G. N. Grant, Yasunori Hayashi, Toru Takumi</p>
Proteome analyses of the postsynaptic density (PSD), a proteinaceous specialization beneath the postsynaptic membrane of excitatory synapses, have identified several thousands of proteins. While proteins with predictable functions have been well studied, functionally uncharacterized proteins are mostly overlooked. In this study, we conducted a comprehensive meta-analysis of 35 PSD proteome datasets, encompassing a total of 5,869 proteins. Employing a ranking methodology, we identified 97 proteins that remain inadequately characterized. From this selection, we focused our detailed analysis on the highest-ranked protein, FAM81A. FAM81A interacts with PSD proteins, including PSD-95, SynGAP, and NMDA receptors, and promotes liquid–liquid phase separation of those proteins in cultured cells or in vitro. Down-regulation of FAM81A in cultured neurons causes a decrease in the size of PSD-95 puncta and the frequency of neuronal firing. Our findings suggest that FAM81A plays a crucial role in facilitating the interaction and assembly of proteins within the PSD, and its presence is important for maintaining normal synaptic function. Additionally, our methodology underscores the necessity for further characterization of numerous synaptic proteins that still lack comprehensive understanding.Pharmacological induction of autophagy reduces inflammation in macrophages by degrading immunoproteasome subunitsJiao ZhouChunxia LiMeng LuGaoyue JiangShanze ChenHuihui LiKefeng Lu10.1371/journal.pbio.30025372024-03-06T14:00:00Z2024-03-06T14:00:00Z<p>by Jiao Zhou, Chunxia Li, Meng Lu, Gaoyue Jiang, Shanze Chen, Huihui Li, Kefeng Lu</p>
Defective autophagy is linked to proinflammatory diseases. However, the mechanisms by which autophagy limits inflammation remain elusive. Here, we found that the pan-FGFR inhibitor LY2874455 efficiently activated autophagy and suppressed expression of proinflammatory factors in macrophages stimulated by lipopolysaccharide (LPS). Multiplex proteomic profiling identified the immunoproteasome, which is a specific isoform of the 20s constitutive proteasome, as a substrate that is degraded by selective autophagy. SQSTM1/p62 was found to be a selective autophagy-related receptor that mediated this degradation. Autophagy deficiency or p62 knockdown blocked the effects of LY2874455, leading to the accumulation of immunoproteasomes and increases in inflammatory reactions. Expression of proinflammatory factors in autophagy-deficient macrophages could be reversed by immunoproteasome inhibitors, confirming the pivotal role of immunoproteasome turnover in the autophagy-mediated suppression on the expression of proinflammatory factors. In mice, LY2874455 protected against LPS-induced acute lung injury and dextran sulfate sodium (DSS)-induced colitis and caused low levels of proinflammatory cytokines and immunoproteasomes. These findings suggested that selective autophagy of the immunoproteasome was a key regulator of signaling via the innate immune system.Viral regulation of organelle membrane contact sitesWilliam A. HofstadterElene TsopurashviliIleana M. Cristea10.1371/journal.pbio.30025292024-03-05T14:00:00Z2024-03-05T14:00:00Z<p>by William A. Hofstadter, Elene Tsopurashvili, Ileana M. Cristea</p>
At the core of organelle functions lies their ability and need to form dynamic organelle–organelle networks that drive intracellular communication and coordination of cellular pathways. These networks are facilitated by membrane contact sites (MCSs) that promote both intra-organelle and inter-organelle communication. Given their multiple functions, MCSs and the proteins that form them are commonly co-opted by viruses during infection to promote viral replication. This Essay discusses mechanisms acquired by diverse human viruses to regulate MCS functions in either proviral processes or host defense. It also examines techniques used for examining MCSs in the context of viral infections.An engineered bacterial symbiont allows noninvasive biosensing of the honey bee gut environmentAudam ChhunSilvia Moriano-GutierrezFlorian ZoppiAmélie CabirolPhilipp EngelYolanda Schaerli10.1371/journal.pbio.30025232024-03-05T14:00:00Z2024-03-05T14:00:00Z<p>by Audam Chhun, Silvia Moriano-Gutierrez, Florian Zoppi, Amélie Cabirol, Philipp Engel, Yolanda Schaerli</p>
The honey bee is a powerful model system to probe host–gut microbiota interactions, and an important pollinator species for natural ecosystems and for agriculture. While bacterial biosensors can provide critical insight into the complex interplay occurring between a host and its associated microbiota, the lack of methods to noninvasively sample the gut content, and the limited genetic tools to engineer symbionts, have so far hindered their development in honey bees. Here, we built a versatile molecular tool kit to genetically modify symbionts and reported for the first time in the honey bee a technique to sample their feces. We reprogrammed the native bee gut bacterium <i>Snodgrassella alvi</i> as a biosensor for IPTG, with engineered cells that stably colonize the gut of honey bees and report exposure to the molecules in a dose-dependent manner through the expression of a fluorescent protein. We showed that fluorescence readout can be measured in the gut tissues or noninvasively in the feces. These tools and techniques will enable rapid building of engineered bacteria to answer fundamental questions in host–gut microbiota research.Cross-frequency coupling in cortico-hippocampal networks supports the maintenance of sequential auditory information in short-term memoryArthur BorderieAnne CaclinJean-Philippe LachauxMarcela Perrone-BertollottiRoxane S. HoyerPhilippe KahaneHélène CatenoixBarbara TillmannPhilippe Albouy10.1371/journal.pbio.30025122024-03-05T14:00:00Z2024-03-05T14:00:00Z<p>by Arthur Borderie, Anne Caclin, Jean-Philippe Lachaux, Marcela Perrone-Bertollotti, Roxane S. Hoyer, Philippe Kahane, Hélène Catenoix, Barbara Tillmann, Philippe Albouy</p>
It has been suggested that cross-frequency coupling in cortico-hippocampal networks enables the maintenance of multiple visuo-spatial items in working memory. However, whether this mechanism acts as a global neural code for memory retention across sensory modalities remains to be demonstrated. Intracranial EEG data were recorded while drug-resistant patients with epilepsy performed a delayed matched-to-sample task with tone sequences. We manipulated task difficulty by varying the memory load and the duration of the silent retention period between the to-be-compared sequences. We show that the strength of theta-gamma phase amplitude coupling in the superior temporal sulcus, the inferior frontal gyrus, the inferior temporal gyrus, and the hippocampus (i) supports the short-term retention of auditory sequences; (ii) decodes correct and incorrect memory trials as revealed by machine learning analysis; and (iii) is positively correlated with individual short-term memory performance. Specifically, we show that successful task performance is associated with consistent phase coupling in these regions across participants, with gamma bursts restricted to specific theta phase ranges corresponding to higher levels of neural excitability. These findings highlight the role of cortico-hippocampal activity in auditory short-term memory and expand our knowledge about the role of cross-frequency coupling as a global biological mechanism for information processing, integration, and memory in the human brain.Cep131-Cep162 and Cby-Fam92 complexes cooperatively maintain Cep290 at the basal body and contribute to ciliogenesis initiationZhimao WuHuicheng ChenYingying ZhangYaru WangQiaoling WangCéline AugièreYanan HouYuejun FuYing PengBénédicte DurandQing Wei10.1371/journal.pbio.30023302024-03-05T14:00:00Z2024-03-05T14:00:00Z<p>by Zhimao Wu, Huicheng Chen, Yingying Zhang, Yaru Wang, Qiaoling Wang, Céline Augière, Yanan Hou, Yuejun Fu, Ying Peng, Bénédicte Durand, Qing Wei</p>
Cilia play critical roles in cell signal transduction and organ development. Defects in cilia function result in a variety of genetic disorders. Cep290 is an evolutionarily conserved ciliopathy protein that bridges the ciliary membrane and axoneme at the basal body (BB) and plays critical roles in the initiation of ciliogenesis and TZ assembly. How Cep290 is maintained at BB and whether axonemal and ciliary membrane localized cues converge to determine the localization of Cep290 remain unknown. Here, we report that the Cep131-Cep162 module near the axoneme and the Cby-Fam92 module close to the membrane synergistically control the BB localization of Cep290 and the subsequent initiation of ciliogenesis in <i>Drosophila</i>. Concurrent deletion of any protein of the Cep131-Cep162 module and of the Cby-Fam92 module leads to a complete loss of Cep290 from BB and blocks ciliogenesis at its initiation stage. Our results reveal that the first step of ciliogenesis strictly depends on cooperative and retroactive interactions between Cep131-Cep162, Cby-Fam92 and Cep290, which may contribute to the complex pathogenesis of Cep290-related ciliopathies.Value-related learning in the olfactory bulb occurs through pathway-dependent perisomatic inhibition of mitral cellsSander LindemanXiaochen FuJanine Kristin ReinertIzumi Fukunaga10.1371/journal.pbio.30025362024-03-01T14:00:00Z2024-03-01T14:00:00Z<p>by Sander Lindeman, Xiaochen Fu, Janine Kristin Reinert, Izumi Fukunaga</p>
Associating values to environmental cues is a critical aspect of learning from experiences, allowing animals to predict and maximise future rewards. Value-related signals in the brain were once considered a property of higher sensory regions, but their wide distribution across many brain regions is increasingly recognised. Here, we investigate how reward-related signals begin to be incorporated, mechanistically, at the earliest stage of olfactory processing, namely, in the olfactory bulb. In head-fixed mice performing Go/No-Go discrimination of closely related olfactory mixtures, rewarded odours evoke widespread inhibition in one class of output neurons, that is, in mitral cells but not tufted cells. The temporal characteristics of this reward-related inhibition suggest it is odour-driven, but it is also context-dependent since it is absent during pseudo-conditioning and pharmacological silencing of the piriform cortex. Further, the reward-related modulation is present in the somata but not in the apical dendritic tuft of mitral cells, suggesting an involvement of circuit components located deep in the olfactory bulb. Depth-resolved imaging from granule cell dendritic gemmules suggests that granule cells that target mitral cells receive a reward-related extrinsic drive. Thus, our study supports the notion that value-related modulation of olfactory signals is a characteristic of olfactory processing in the primary olfactory area and narrows down the possible underlying mechanisms to deeper circuit components that contact mitral cells perisomatically.Structural and functional characterization of AfsR, an SARP family transcriptional activator of antibiotic biosynthesis in <i>Streptomyces</i>Yiqun WangXu YangFeng YuZixin DengShuangjun LinJianting Zheng10.1371/journal.pbio.30025282024-03-01T14:00:00Z2024-03-01T14:00:00Z<p>by Yiqun Wang, Xu Yang, Feng Yu, Zixin Deng, Shuangjun Lin, Jianting Zheng</p>
<i>Streptomyces</i> antibiotic regulatory proteins (SARPs) are widely distributed activators of antibiotic biosynthesis. <i>Streptomyces coelicolor</i> AfsR is an SARP regulator with an additional nucleotide-binding oligomerization domain (NOD) and a tetratricopeptide repeat (TPR) domain. Here, we present cryo-electron microscopy (cryo-EM) structures and in vitro assays to demonstrate how the SARP domain activates transcription and how it is modulated by NOD and TPR domains. The structures of transcription initiation complexes (TICs) show that the SARP domain forms a side-by-side dimer to simultaneously engage the <i>afs box</i> overlapping the −35 element and the σ<sup>HrdB</sup> region 4 (R4), resembling a sigma adaptation mechanism. The SARP extensively interacts with the subunits of the RNA polymerase (RNAP) core enzyme including the β-flap tip helix (FTH), the β′ zinc-binding domain (ZBD), and the highly flexible C-terminal domain of the α subunit (αCTD). Transcription assays of full-length AfsR and truncated proteins reveal the inhibitory effect of NOD and TPR on SARP transcription activation, which can be eliminated by ATP binding. In vitro phosphorylation hardly affects transcription activation of AfsR, but counteracts the disinhibition of ATP binding. Overall, our results present a detailed molecular view of how AfsR serves to activate transcription.An adapted MS2-MCP system to visualize endogenous cytoplasmic mRNA with live imaging in <i>Caenorhabditis elegans</i>Cristina TocchiniSusan E. Mango10.1371/journal.pbio.30025262024-03-01T14:00:00Z2024-03-01T14:00:00Z<p>by Cristina Tocchini, Susan E. Mango</p>
Live imaging of RNA molecules constitutes an invaluable means to track the dynamics of mRNAs, but live imaging in <i>Caenorhabditis elegans</i> has been difficult to achieve. Endogenous transcripts have been observed in nuclei, but endogenous mRNAs have not been detected in the cytoplasm, and functional mRNAs have not been generated. Here, we have adapted live imaging methods to visualize mRNA in embryonic cells. We have tagged endogenous transcripts with MS2 hairpins in the 3′ untranslated region (UTR) and visualized them after adjusting MS2 Coat Protein (MCP) expression. A reduced number of these transcripts accumulates in the cytoplasm, leading to loss-of-function phenotypes. In addition, during epithelial morphogenesis, MS2-tagged mRNAs for <i>dlg-1</i> fail to associate with the adherens junction, as observed for untagged, endogenous mRNAs. These defects are reversed by inactivating the nonsense-mediated decay pathway. RNA accumulates in the cytoplasm, mutant phenotypes are rescued, and <i>dlg-1</i> RNA associates with the adherens junction. These data suggest that MS2 repeats can induce the degradation of endogenous RNAs and alter their cytoplasmic distribution. Although our focus is RNAs expressed in epithelial cells during morphogenesis, we find that this method can be applied to other cell types and stages.Ebola virus VP35 interacts non-covalently with ubiquitin chains to promote viral replicationCarlos A. Rodríguez-SalazarSarah van TolOlivier MailhotMaria Gonzalez-OrozcoGabriel T. GaldinoAbbey N. WarrenNatalia TeruelPadmanava BeheraKazi Sabrina AfreenLihong ZhangTerry L. JuelichJennifer K. SmithMaría Inés ZylberAlexander N. FreibergRafael J. NajmanovichMaria I. GiraldoRicardo Rajsbaum10.1371/journal.pbio.30025442024-02-29T14:00:00Z2024-02-29T14:00:00Z<p>by Carlos A. Rodríguez-Salazar, Sarah van Tol, Olivier Mailhot, Maria Gonzalez-Orozco, Gabriel T. Galdino, Abbey N. Warren, Natalia Teruel, Padmanava Behera, Kazi Sabrina Afreen, Lihong Zhang, Terry L. Juelich, Jennifer K. Smith, María Inés Zylber, Alexander N. Freiberg, Rafael J. Najmanovich, Maria I. Giraldo, Ricardo Rajsbaum</p>
Ebolavirus (EBOV) belongs to a family of highly pathogenic viruses that cause severe hemorrhagic fever in humans. EBOV replication requires the activity of the viral polymerase complex, which includes the cofactor and Interferon antagonist VP35. We previously showed that the covalent ubiquitination of VP35 promotes virus replication by regulating interactions with the polymerase complex. In addition, VP35 can also interact non-covalently with ubiquitin (Ub); however, the function of this interaction is unknown. Here, we report that VP35 interacts with free (unanchored) K63-linked polyUb chains. Ectopic expression of Isopeptidase T (USP5), which is known to degrade unanchored polyUb chains, reduced VP35 association with Ub and correlated with diminished polymerase activity in a minigenome assay. Using computational methods, we modeled the VP35-Ub non-covalent interacting complex, identified the VP35-Ub interacting surface, and tested mutations to validate the interface. Docking simulations identified chemical compounds that can block VP35-Ub interactions leading to reduced viral polymerase activity. Treatment with the compounds reduced replication of infectious EBOV in cells and in vivo in a mouse model. In conclusion, we identified a novel role of unanchored polyUb in regulating Ebola virus polymerase function and discovered compounds that have promising anti-Ebola virus activity.New twists in the evolution of retinal direction selectivityTakeshi YoshimatsuTom Baden10.1371/journal.pbio.30025382024-02-29T14:00:00Z2024-02-29T14:00:00Z<p>by Takeshi Yoshimatsu, Tom Baden</p>
In mammals, starburst amacrine cells are centrally involved in motion vision and a new study in PLOS Biology, by Yan and colleagues finds that zebrafish have them, too. They coexist with a second pair of starburst-like neurons, but neither appears to be strongly motion selective.
In mammals, starburst amacrine cells are centrally involved in motion vision. This primer explores the implications of a PLOS Biology study reporting that zebrafish have amacrine cells, too, and revealing that these cells coexist with a second pair of starburst-like neurons, but neither appears to be strongly motion selective.Ultrastructural insights into the microsporidian infection apparatus reveal the kinetics and morphological transitions of polar tube and cargo during host cell invasionHimanshu SharmaNathan JespersenKai EhrenbolgerLars-Anders CarlsonJonas Barandun10.1371/journal.pbio.30025332024-02-29T14:00:00Z2024-02-29T14:00:00Z<p>by Himanshu Sharma, Nathan Jespersen, Kai Ehrenbolger, Lars-Anders Carlson, Jonas Barandun</p>
During host cell invasion, microsporidian spores translocate their entire cytoplasmic content through a thin, hollow superstructure known as the polar tube. To achieve this, the polar tube transitions from a compact spring-like state inside the environmental spore to a long needle-like tube capable of long-range sporoplasm delivery. The unique mechanical properties of the building blocks of the polar tube allow for an explosive transition from compact to extended state and support the rapid cargo translocation process. The molecular and structural factors enabling this ultrafast process and the structural changes during cargo delivery are unknown. Here, we employ light microscopy and in situ cryo-electron tomography to visualize multiple ultrastructural states of the <i>Vairimorpha necatrix</i> polar tube, allowing us to evaluate the kinetics of its germination and characterize the underlying morphological transitions. We describe a cargo-filled state with a unique ordered arrangement of microsporidian ribosomes, which cluster along the thin tube wall, and an empty post-translocation state with a reduced diameter but a thicker wall. Together with a proteomic analysis of endogenously affinity-purified polar tubes, our work provides comprehensive data on the infection apparatus of microsporidia and uncovers new aspects of ribosome regulation and transport.