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SUMMARY Proper function in a bacterial cell relies on intrinsic cell size regulation. The molecular mechanisms underlying how bacteria maintain their cell size remain unclear. The conserved regulator DnaA, the initiator of chromosome replication, is associated to size regulation by controlling the number of origins of replication (oriC) per cell. In this study, we identify and characterize a new mechanism in which DnaA modulates cell size independently oforiC-copy number. By altering the levels of DnaA without impacting chromosome replication, we demonstrate that DnaA’s activity as a transcription factor can slow down cell elongation rate resulting in cells that are ∼20% smaller. We identify the peptidoglycan biosynthetic enzyme MurD as a key player of cell size regulation inCaulobacter crescentusand in the evolutionarily distant bacteriumEscherichia coli. Collectively, our findings provide mechanistic insights to the complex regulation of cell size in bacteria. 

ABSTRACT Many microscopic images and simulations of cells give results in different kinds of formats, making it difficult for people lacking computational skills to visualize and interact with them. Minecraft—known for its three-dimensional, open-world, voxel-based environment—offers a unique solution by allowing the direct insertion of voxel-based cellular structures from light microscopy and simulations into its worlds without modification. This integration enables Minecraft players to explore the ultrastructure of cells in a highly immersive and interactive environment. Here, we demonstrate several workflows that can convert images and simulation results into Minecraft worlds. Using the workflows, students can easily import and interact with a variety of cellular content, including bacteria, yeast, and cancer cells. This approach not only opens new avenues for science education but also demonstrates the potential of combining scientific visualization with interactive gaming platforms for facilitating research and improving appreciation of cellular structure for a broad audience. 

Abstract In addition to regulating the actin cytoskeleton, Cofilin also senses and responds to environmental stress. Cofilin can promote cell survival or death depending on context. Yet, many aspects of Cofilin’s role in survival need clarification. Here, we show that exposing earlyDrosophilaembryos to mild heat stress (32°C) induces a Cofilin-mediated Actin Stress Response and upregulation of heat- and ER-stress response genes. However, these responses do not alleviate the negative impacts of heat exposure. Instead, heat stressed embryos show downregulation of hundreds of developmental genes, including determinants of the embryonic body plan, and are less likely to hatch as larvae and adults. Remarkably, reducing Cofilin dosage blunts induction of all stress response pathways, mitigates downregulation of developmental genes, and completely rescues survival. Thus, Cofilin intersects with multiple stress response pathways, and modulates the transcriptomic response to heat stress. Strikingly, Cofilin knockdown emerges as a potent pro-survival manipulation for embryos. 

Abstract Hypoxia-induced alternative splicing (AS) regulates tumor progression and metastasis. Little is known about how such AS is controlled and whether higher-order genome and nuclear domain (ND) organizations dictate these processes. We observe that hypoxia-responsive alternatively spliced genes position near nuclear speckle (NS), the ND that enhances splicing efficiency. NS-resident MALAT1 long noncoding RNA, induced in response to hypoxia, regulates hypoxia-responsive AS. MALAT1 achieves this by organizing the SR-family of splicing factor, SRSF1, near NS and regulating the binding of SRSF1 to pre-mRNAs. Mechanistically, MALAT1 enhances the recruitment of SRSF1 to elongating RNA polymerase II (pol II) by promoting the formation of phase-separated condensates of SRSF1, which are preferentially recognized by pol II. During hypoxia, MALAT1 regulates spatially organized AS by establishing a threshold SRSF1 concentration near NSs, potentially by forming condensates, critical for pol II-mediated recruitment of SRSF1 to pre-mRNAs. 

Abstract Data integration is a powerful tool for facilitating a comprehensive and generalizable understanding of microbial communities and their association with outcomes of interest. However, integrating data sets from different studies remains a challenging problem because of severe batch effects, unobserved confounding variables, and high heterogeneity across data sets. We propose a new data integration method called MetaDICT, which initially estimates the batch effects by weighting methods in causal inference literature and then refines the estimation via a novel shared dictionary learning. Compared with existing methods, MetaDICT can better avoid the overcorrection of batch effects and preserve biological variation when there exist unobserved confounding variables or data sets are highly heterogeneous across studies. Furthermore, MetaDICT can generate comparable embedding at both taxa and sample levels that can be used to unravel the hidden structure of the integrated data and improve the integrative analysis. Applications to synthetic and real microbiome data sets demonstrate the robustness and effectiveness of MetaDICT in integrative analysis. Using MetaDICT, we characterize microbial interaction, identify generalizable microbial signatures, and enhance the accuracy of disease prediction in an integrative analysis of colorectal cancer metagenomics studies. 

Abstract In eukaryotic cells, transcription, translation, and mRNA degradation occur in distinct subcellular regions. How these mRNA processes are organized in bacteria, without employing membrane-bound compartments, remains unclear. Here, we present generalizable principles underlying coordination between these processes in bacteria. InEscherichia coli, we found that co-transcriptional degradation is rare for mRNAs except for those encoding inner membrane proteins, due to membrane localization of the main ribonuclease, RNase E. We further found, by varying ribosome binding sequences, that translation affects mRNA stability not because ribosomes protect mRNA from degradation, but because low translation leads to premature transcription termination in the absence of transcription-translation coupling. Extending our analyses toBacillus subtilisandCaulobacter crescentus, we established subcellular localization of RNase E (or its homolog) and premature transcription termination in the absence of transcription-translation coupling as key determinants that explain differences in transcriptional and translational coupling to mRNA degradation across genes and species. 

Abstract Despite a vaccine, hepatitis B virus (HBV) remains a world-wide source of infections and deaths. We develop a whole-cell computational platform combining spatial and kinetic models describing the infection cycle of HBV in a hepatocyte host. We simulate key parts of the infection cycle with this whole-cell platform for 10 min of biological time, to predict infection progression, map out virus-host and virus-drug interactions. We find that starting from an established infection, decreasing the copy number of the viral envelope proteins shifts the dominant infection pathway from capsid secretion to re-importing the capsids into the nucleus, resulting in more nuclear-localized viral covalently closed circular DNA (cccDNA) and boosting transcription. This scenario can mimic the consequence of drugs designed to manipulate viral gene expression. Mutating capsid proteins facilitates capsid destabilization and disassembly at nuclear pore complexes, resulting in an increase in cccDNA copy number. However, excessive destabilization leads to premature cytoplasmic disassembly and does not increase the cccDNA counts. Finally, our simulations can predict the best drug dosage and its administration timing to reduce the cccDNA counts. Our adaptable computational platform can be parameterized to study other viruses and identify the most central viral pathways that can be targeted by drugs. 

Abstract Necroptosis is a form of inflammatory lytic cell death involving active cytokine production and plasma membrane rupture. Progression of necroptosis is tightly regulated in time and space, and its signaling outcomes can shape the local inflammatory environment of cells and tissues. Pharmacological induction of necroptosis is well established, but the diffusive nature of chemical death inducers makes it challenging to study cell‐cell communication precisely during necroptosis. Receptor‐interacting protein kinase 3, or RIPK3, is a crucial signaling component of necroptosis, acting as a crucial signaling node for both canonical and non‐canonical necroptosis. RIPK3 oligomerization is crucial to the formation of the necrosome, which regulates plasma membrane rupture and cytokine production. Commonly used necroptosis inducers can activate multiple downstream signaling pathways, confounding the signaling outcomes of RIPK3‐mediated necroptosis. Opsin‐free optogenetic techniques may provide an alternative strategy to address this issue. Optogenetics uses light‐sensitive protein‐protein interaction to modulate cell signaling. Compared to chemical‐based approaches, optogenetic strategies allow for spatiotemporal modulation of signal transduction in live cells and animals. We developed an optogenetic system that allows for ligand‐free optical control of RIPK3 oligomerization and necroptosis. This article describes the sample preparation, experimental setup, and optimization required to achieve robust optogenetic induction of RIPK3‐mediated necroptosis in colorectal HT‐29 cells. We expect that this optogenetic system could provide valuable insights into the dynamic nature of lytic cell death. © 2024 The Authors. Current Protocols published by Wiley Periodicals LLC. Basic Protocol 1: Production of lentivirus encoding the optogenetic RIPK3 system Support Protocol: Quantification of the titer of lentivirus Basic Protocol 2: Culturing, chemical transfection, and lentivirus transduction of HT‐29 cells Basic Protocol 3: Optimization of optogenetic stimulation conditions Basic Protocol 4: Time‐stamped live‐cell imaging of HT‐29 lytic cell death Basic Protocol 5: Quantification of HT‐29 lytic cell death 

Fourier-transform infrared spectroscopy (FTIR) is a powerful analytical method not only for the chemical identification of solid, liquid, and gas species but also for the quantification of their concentration. However, the chemical quantification capability of FTIR is significantly hindered when the analyte is surrounded by a strong IR absorbing medium, such as liquid solutions. To overcome this limit, here we develop an IR fiber microprobe that can be inserted into a liquid medium and obtain full FTIR spectra at points of interest. To benchmark this endoscopic FTIR method, we insert the microprobe into bulk water covering a ZnSe substrate and measure the IR transmittance of water as a function of the probe–substrate distance. The obtained vibrational modes, overall transmittance vs z profiles, quantitative absorption coefficients, and micro z-section IR transmittance spectra are all consistent with the standard IR absorption properties of water. The results pave the way for endoscopic chemical profiling inside bulk liquid solutions, promising for applications in many biological, chemical, and electrochemical systems. 

Enzyme–enzyme interactions are fundamental to the function of cells. Their atomistic mechanisms remain elusive mainly due to limitations of in-cell measurements. We address this challenge by atomistically modeling, for a total of ≈80 μs, a slice of the human cell cytoplasm that includes three successive enzymes along the glycolytic pathway: glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (PGK), and phosphoglycerate mutase (PGM). We tested the model for nonspecific protein stickiness, an artifact of current atomistic force fields in crowded environments. The simulations reveal that the human enzymes co-organize in-cell into transient submetabolon complexes, consistent with previous experimental results. Our data both reiterate known specificity between GAPDH and PGK and reveal extensive direct interactions between GAPDH and PGM. Our simulations further reveal, through force field benchmarking, the critical role of protein solvation in facilitating these enzyme–enzyme interactions. Transient interenzyme interactions with μs lifetime occur repeatedly in our simulations via specific sticky protein surface patches, with interactions often mediated by charged patch residues. Some of the residues that interact frequently with one another lie in or near the active site of the enzymes. We show that some of these patches correspond to a general mode to interact with several partners for promiscuous enzymes like GAPDH. We further show that the non-native yeast PGK is stickier than human PGK in our human cytoplasm model, supporting the idea of evolutionary pressure to reduce sticking. Our cytoplasm modeling paves the way toward capturing the atomistic dynamics of an entire enzymatic pathway in-cell.