Once described as mere “bags of enzymes,” bacterial cells are in fact highly organized, with many macromolecules exhibiting nonuniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid–liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in Escherichia coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrient-rich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components. Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and demonstrates that this process can serve as a mechanism for intracellular organization in prokaryotes and eukaryotes alike.

Once described as mere "bags of enzymes", bacterial cells are in fact highly organized, with many macromolecules exhibiting non-uniform localization patterns. Yet the physical and biochemical mechanisms that govern this spatial heterogeneity remain largely unknown. Here, we identify liquid-liquid phase separation (LLPS) as a mechanism for organizing clusters of RNA polymerase (RNAP) in E. coli. Using fluorescence imaging, we show that RNAP quickly transitions from a dispersed to clustered localization pattern as cells enter log phase in nutrientrich media. RNAP clusters are sensitive to hexanediol, a chemical that dissolves liquid-like compartments in eukaryotic cells. In addition, we find that the transcription antitermination factor NusA forms droplets in vitro and in vivo, suggesting that it may nucleate RNAP clusters. Finally, we use single-molecule tracking to characterize the dynamics of cluster components.Our results indicate that RNAP and NusA molecules move inside clusters, with mobilities faster than a DNA locus but slower than bulk diffusion through the nucleoid. We conclude that RNAP clusters are biomolecular condensates that assemble through LLPS. This work provides direct evidence for LLPS in bacteria and suggests that this process serves as a universal mechanism for intracellular organization across the tree of life. SignificanceBacterial cells are small and were long thought to have little to no internal structure. However, advances in microscopy have revealed that bacteria do indeed contain subcellular compartments. But how these compartments form has remained a mystery. Recent progress in larger, more complex eukaryotic cells has identified a novel mechanism for intracellular organization known as liquid-liquid phase separation. This process causes certain types of molecules to concentrate within distinct compartments inside the cell. Here, we demonstrate that the same process also occurs in bacteria. This work, together with a growing body of literature, suggests that liquidliquid phase separation is a universal mechanism for intracellular organization that extends across the tree of life.

Traditional arc-based light sources and Light Emitting Diodes (LEDs) are described, and the pros and cons of these sources with respect to fluorescence microscopy are discussed. For multi-color applications, arc-based light sources offer white light ranging from the ultraviolet (UV) to the infrared (IR), while LEDs come in a range of colors spanning the same wavelengths. The power of traditional arc-based sources is controlled with neutral density (ND) filters, reducing power across the entire range of wavelengths, while LED-based sources can be controlled directly by modulating current passing through the electronics. Similarly, arc-based sources use physical shutters to control sample exposure to light in a range of tens to hundreds of milliseconds (ms), while LEDs can be turned ON/OFF electronically in <1 ms. The complexity of comparing and measuring light power on the sample, due to normalization of available light source spectra and complex power measurements, is discussed. The superiority of LEDs for stability of light power output is covered. Direct coupling of light sources to the microscope is more cost effective and leads to higher available light power. Various options for setting up multi-color imaging, including high-speed imaging with multiple LEDs and a triple cube, are described. A brief introduction to lasers, with suggested further reading, is included in this article. Finally, the smaller environmental footprint of LEDs relative to arc-based light sources is highlighted.

CHD4 is a catalytic subunit of the NuRD (nucleosome remodeling and deacetylase) complex essential in transcriptional regulation, chromatin assembly and DNA damage repair. CHD4 contains tandem plant homeodomain (PHD) fingers connected by a short linker, the biological function of which remains unclear. Here we explore the combinatorial action of the CHD4 PHD1/2 fingers and detail the molecular basis of their association with chromatin. We found that the inter-domain organization of PHD1/2 leads to specific targeting of a single nucleosome in a multivalent manner, concomitantly engaging both of the histone H3 tails. This robust synergistic interaction displaces HP1g from pericentric sites, inducing changes in chromatin structure and dispersion of the heterochromatic marks H3K9me3 and H4K20me3. We demonstrate that recognition of the histone H3 tails by the PHD fingers is required for repressive activity of the CHD4/NuRD complex. Together, our data elucidate the molecular mechanism of multivalent association of the PHD fingers with chromatin and reveal their critical role in the regulation of CHD4 functions.

In the above-referenced article, Figure 2 and panels A and B of Figure 3 were replaced with new versions in which the readability was improved and the labels on the face of the figure were updated by the author.

BackgroundImmune checkpoint inhibitors (ICI) have changed the landscape of treatment for many cancers. However, most cancer clinical trials for ICI excluded patients with pre-existing autoimmune disease (PAD). Efficacy and safety data on the use of ICI in patients with rheumatic PAD (Rh-PAD) is limited to retrospective case series and reports, and many do not differentiate between Rh-PAD and non-rheumatic PAD. In addition, little is known about optimal use of concurrent immunosuppression and its impact on PAD flares and development of de novo irAE. There is some data that patients on immunosuppression at baseline are at risk for poorer tumor outcomes [1].ObjectivesTo explore the safety and efficacy of ICI in patients with Rh-PAD and to determine if immunosuppression at baseline impacts risk of PAD flare, de-novo irAE and early tumor outcomes using data from the CanRIO prospective cohort.MethodsThe CanRIO cohort includes adult patients with Rh-PAD treated with immune checkpoint inhibitors (ICI, including CTLA-4, PD-1 or PDL-1 inhibitors), recruited across 9 Canadian academic sites and followed prospectively at regular intervals as per a standardized study protocol. In this study, we evaluated patients with Rh-PAD who received at least one dose of ICI.ResultsForty patients with Rh-PADs were recruited into the CanRIO prospective cohort between Jan 2020 and Nov 2022, including 14 (35%) with rheumatoid arthritis, 11 (27.5%) psoriatic arthritis, 7 (17.5%) polymyalgia rheumatica, 5 (12.5%) ankylosing spondylitis and 7 (17.5%) other. The most frequently observed cancers were melanoma 14 (35%) and lung cancer 10 (25%), with stage 3 and stage 4 disease each making up 37.5% of all cases. 25 patients (62.5%) were on baseline immunosuppression prior to ICI start. Anti-PD-1 therapy was most common (45%), followed by combination ICI therapy (22.5%), with a median exposure of 2 months (IQR: 0.5-3.0). Over a median (IQR) follow-up of 3.0 (1.0–6.0) months, 55% experienced a de-novo irAE (68.2% with rh-irAE; of which 46.7% were on baseline immunosuppression), 25% had Rh-PAD flare, of which 60% were on baseline immunosuppression. Of these, 40% were severe (of which 50% on baseline immunosuppression); 70% required corticosteroid treatment; and 40% required a change in immunosuppression. ICI was continued in 50%, transiently held or delayed in 40% and stopped in only 10%. Of the 9 patients on baseline immunosuppression who had been re-staged, 8 had a favorable tumor response (i.e., stabilization, partial or complete response) and only 1 had disease progression.ConclusionEarly data from patients with Rh-PAD in the CanRIO multi-center prospective cohort suggests that (1) ICI can be effective for cancer treatment in patients with Rh-PAD and should be offered as indicated; (2) flares of Rh-PAD are common but can be managed with corticosteroids or escalation of immunosuppression; (3) baseline immunosuppression did not significantly change rate of de-novo irAE or rate/severity of PAD flare (3) most patients with Rh-PADs had good tumour response despite baseline immunosuppression.Reference[1]Tison A, Quéré G, Misery L, Funck-Brentano E, Danlos FX, Routier E, et al. Safety and efficacy of immune checkpoint inhibitors in patients with cancer and preexisting autoimmune disease: A nationwide, Multicenter Cohort Study. Arthritis & Rheumatology. 2019;71(12):2100–11.AcknowledgementsWe would like to thank Bristol-Myers Squibb, Merck, and CIORA (Canadian Initiative for Outcomes in Rheumatology cAre) for supporting this study. This content is solely the responsibility of the authors and does not necessarily represent the official views of the sponsors listed here. This research was conducted as part of the Canadian Research Group of Rheumatology in Immuno-Oncology (CanRIO).Disclosure of InterestsLourdes Gonzalez Arreola: None declared, Jeremiah Tan: None declared, Alexandra Ladouceur: None declared, Marie Hudson Consultant of: Consulting fees: Boehringer Ingelheim, Grant/research support from: Unrestricted research grants: Bristol-Myers Squibb, Boehringer Ingelheim; Clinical studies: Astra-Zeneca, Carrie Ye: None declared, Janet Roberts: None declared, Aurore Fifi-Mah: None declared, May Choi: None declared, Sabrina Hoa: None declared, C. Thomas Appleton: None declared, Janet Pope: None declared, Nancy Maltez: None declared, Shahin Jamal: None declared.