Biology subjects

Pradhan, B.

Publications and source records attributed to Pradhan, B..

At least 19 recordsLinked to original sources

bioRxiv2026

Two polymorphs of an independent amyloid core in medin's C-terminal region.

Medin, a 50-residue fragment of lactadherin (MFG-E8), forms aortic medial amyloid, the most common localized amyloid in humans, and co-deposits with vascular amyloid-{beta} (A{beta}) in Alzheimer's disease. In the full-length medin fibril the N-terminal half forms the entire protofilament interface, yet the strongest predicted amyloidogenic determinants lie in the C-terminal region. Here we show that this region aggregates on its own. The isolated C-terminal aggregation-prone region (medin C-APR, medin 31-53) begins to aggregate with the same lag time as full-length medin and assembles into two coexisting polymorphs, determined here by cryo-EM at 2.71 and 2.91 [A]. Both are built from the same two-stranded {beta}-arch and a pseudo-21 protofilament pair closed by an aromatic-aliphatic zipper, and differ only in how many accessory chains the exposed hydrophobic face recruits. The medin C-terminus therefore carries an amyloid core of its own, unrelated to that of the full-length fibril: medin has more than one aggregation-prone region, and the structural contribution of each region depends on sequence and assembly context.

biochemistry↗

bioRxiv2026

An integrated database of ion channels

Ion channels play critical roles in a wide range of physiological processes, including the propagation of nerve impulses, insulin secretion and regulation of heart rate. Alterations in ion-channels function or expression can contribute to the development of various diseases, including cancers, genetic disorders, and infectious diseases. Ion channels represent the second-largest class of membrane proteins considered potential targets for therapeutic intervention. However, a comprehensive data resource integrating information on human ion channels and their associations with diseases has been limited. Therefore, the Integrated Database of Ion Channels (IDIC) was developed, comprising information on 493 human ion channels. The database contains a broad range of ion-channel annotations, including gene and protein information, structural details, protein signature sequences, protein domains, nuclear localization signals, and signal peptides. It also provides cross-references to several established biological resources, including the Human Genome Organization Gene Nomenclature Committee (HGNC), National Center for Biotechnology Information (NCBI) Gene, Vertebrate Genome Annotation (VEGA), Ensembl, and the Protein Data Bank (PDB). Furthermore, IDIC incorporates genomic variations identified in various tumors and disease associations involving ion channels, which have been systematically curated from published literature. Through concise search methods and user-friendly browsing functionalities, IDIC serves as an integrated resource for exploring human ion channels and investigating their structural, functional, genetic, and disease-related characteristics. Thus, the database can facilitate a better understanding of ion channels and their potential involvement in diverse human diseases.

bioinformatics↗

bioRxiv2026

Pre-catalytic activation pathway of the Smc5/6 complex

The Smc5/6 complex (Smc5/6) preserves genome stability through ATP-dependent DNA organization, but the structural details that prime the complex for productive DNA engagement remain unknown. Here, we combine cryo-electron microscopy and conformational heterogeneity analysis to define the pre-catalytic activation pathway of Smc5/6. We show that in the absence of ATP and DNA, Smc5/6 exists as a heterogeneous conformational ensemble composed predominantly of two conformations termed Bridge and Cross. In the presence of DNA alone, Smc5/6 adopts a Juxtaposed conformation that brings the ATPase heads into proximity, aligning them for nucleotide engagement. With both ATP and DNA associated, Smc5/6 establishes an Engaged architecture, within which DNA stabilizes its head-associated binding cavity through local remodeling of the complex. Conformational heterogeneity analyses suggest a continuous structural transition between the Cross and Juxtaposed states, while anisotropic network modeling identifies the Bridge-Cross-Juxtaposed-Engaged sequence as the mechanically favored pathway connecting the four experimentally observed conformations. Together, these findings support a pre-catalytic conformational pathway for Smc5/6 activation and provide a structural framework for understanding how conformational heterogeneity gives rise to an activation-competent motor state.

biochemistry↗

bioRxiv2026

High-resolution CRISPR/Cas9 screens identify PAK2 as a suppressor of macrophage proliferation

Macrophage abundance is regulated by pathways controlling survival and proliferation, yet the genetic determinants of macrophage fitness remain poorly understood. Here, we performed replicate genome-wide CRISPR/Cas9 screens in primary murine bone marrow-derived macrophages (BMDMs) to identify positive and negative regulators of macrophage fitness. Validity of the screens is supported by finding previously characterized core essential genes such as those encoding translation machinery, while disruption of established tumor suppressors increased BMDM representation in the culture. As expected, Csf1r encoding the macrophage growth factor emerged as a positive regulator of macrophage fitness. Unexpectedly, Pak2, encoding p21-activated kinase 2 (PAK2), emerged as a negative regulator of macrophage fitness. Targeted Pak2 disruption increased BMDM accumulation and was associated with elevated cyclin-D1 expression, identifying PAK2 as a suppressor of macrophage proliferation. PAK2 also localized to CSF1-induced actin-rich membrane ruffles, yet its depletion did not impair ruffle formation. Instead, PAK2-depleted macrophages exhibited persistent F-actin-rich ruffles, larger macropinosomes, and increased fluid-phase uptake. These changes were accompanied by altered LIMK/cofilin signaling and persistent cofilin localization at macropinocytic structures. Phosphoproteomic analysis further identified reduced phosphorylation of proteins associated with cytoskeletal regulation, phosphoinositide signaling, and endosomal trafficking. Together, these findings identify PAK2 as a context-dependent regulator that restrains macrophage proliferation and membrane-remodeling activity and demonstrate that PAK2 has markedly different fitness functions in primary macrophages than previously identified in several transformed cell types.

cell biology↗

bioRxiv2026

PI(3)P Signaling by VPS34 Complex II Orchestrates Macropinocytosis

Macrophage macropinocytosis contributes to wound healing, antigen presentation, and resolution of inflammation. Macropinocytosis also facilitates nutrient uptake and growth in macrophages, T cells, and cancer cells. Here, CRISPR/Cas9 whole-genome screens in murine bone-marrow derived macrophages (BMDM) identified genes regulating unstimulated, PMA-, and CSF1-stimulated uptake of the fluorescent pinocytosis solute tracer, Lucifer yellow. UVRAG and other members of VPS34 complex II (VPS34-II), which catalyze PI(3)P formation from phosphatidylinositol, were identified as positive regulators of macropinocytosis. Targeted gene disruption of Uvrag and Pik3r3 revealed that VPS34-II is required for efficient macropinocytosis with Uvrag-disrupted BMDM having fewer but larger macropinosomes. In contrast, depletion of ATG14, a unique component of VPS34 complex I, increased solute uptake and the number of macropinosomes. Live-cell imagining of macrophages expressing 2xFYVE-fluorescent protein fusions showed PI(3)P present on the plasma membrane, nascent macropinosomes, and endosomes. The presence of PI(3)P on the plasma membrane prior to macropinocytic cup closure, indicates a novel role on the plasma membrane. Quantitative imaging of fixed cells shows a decrease in concentration of PI(3)P in Uvrag-disrupted BMDMs while increase in concentration of PI(3)P in Atg14-disrupted BMDM compared to WT BMDMs. Treatment with the VPS34 inhibitor SAR-405 acutely decreased macropinocytosis but maintained AKT phosphorylation suggesting class I PI3K activity and PI(3,4,5)P3 production are independent of class III PI3K activity. These results suggest that PI(3)P is a key phosphoinositide governing macropinocytosis at the plasma membrane and that it is primarily formed via direct phosphorylation of PI rather than via the sequential dephosphorylation of PIP3.

cell biology↗

bioRxiv2026

Probing intracellular physical environments by rotational and translational single-particle tracking

Single-particle tracking is widely used to probe nanoscale dynamics in biological systems, yet most approaches rely exclusively on translational motion, overlooking rotational dynamics that offer complementary information about the local physical environment. Here, we present a simultaneous rotational and translational single-particle tracking approach using a vortex-engineered point spread function in a single detection channel. We validate this approach with static and freely diffusing nanorods, demonstrating accurate orientation recovery and quantitative agreement with theoretical predictions of rotational diffusion. Using a biomimetic lipid bilayer system, we show that translational and rotational diffusion exhibit distinct sensitivities to environmental perturbations, confirming that these two modalities capture complementary local environment information. Applying this framework to living HeLa cells, we show that combined translational and rotational diffusion signatures define distinct biophysical fingerprints of cytoplasmic and endocytic compartments and reveal compartment-specific responses to metabolic perturbation. Finally, time-resolved analysis of individual endocytic compartments uncovers dynamic changes in the local physical environment that are inaccessible to conventional translational tracking. By coupling translational and rotational readouts, this framework opens a new dimension for probing the physical organization and dynamics of living systems at the nanoscale.

biophysics↗

bioRxiv2026

Evidence of residual Brugia malayi infection in TAS-III cleared areas of coastal Odisha: Findings from Molecular Xenomonitoring

BackgroundLymphatic filariasis (LF), caused by Wuchereria (W) bancrofti, Brugia malayi and Brugia (B) timori, remains a major public health problem known for its disfiguring and debilitating effects despite notable progress under the Global Program to Eliminate Lymphatic Filariasis (GPELF). Following recent national directives (2024) restructuring implementation units (IUs) to the block level, all TAS-cleared blocks now require re-evaluation using night blood surveys (NBS) or filarial test strip (FTS) kits. Molecular Xenomonitoring (MX) is recommended as a post-validation surveillance method for detecting active LF transmission in vectors. Therefore, an exploratory study was conducted in two coastal districts of Odisha, namely Jagatsinghpur and Puri, which cleared TAS-III, to investigate the presence of B. malayi and W. bancrofti infections in filaria-transmitting vectors. Methodology/Principal FindingsAdult female mosquitoes were collected from three villages across Jagatsinghpur and Puri districts during March-April 2025. Species were identified morphologically, segregated and pooled. The pooled samples were screened for the presence of filarial DNA using species-specific PCR assays. Of 1,518 collected adult female mosquitoes, 22.6% were Culex quinquefasciatus, 18.3% were Mansonia (Mn) annulifera, and 4.3% were Mansonia uniformis. Among 33 pools screened, six pools were positive for B. malayi DNA (18.18% pool positivity; infection rate 2.88%, 95% CI: 1.23-5.74), in Mn. annulifera, whereas W. bancrofti was not detected. Conclusions/SignificanceThe study provides the first molecular confirmation of B. malayi infection in Mn. annulifera, indicating a persistent Brugian transmission in Odisha even after the implementation of mass drug administration. The Study highlights MX as a sensitive, non-invasive surveillance tool for detecting residual infection in post-MDA and post-TAS settings. The study underscores the need for a standardized MX protocol and integration with entomological monitoring to sustain national LF elimination targets by 2027. Author SummaryLymphatic filariasis is a mosquito-borne disease that can cause severe, disfiguring swelling of the limbs and other lasting disabilities, and it remains a serious public health problem in India. National programmes in India are actively working to eliminate it by giving preventive medicines to communities, conducting surveys to confirm that transmission has ceased, and once an area passes, this treatment is withdrawn. However, it is not always clear whether the parasite has truly disappeared or is still lingering at very low levels. In this study, we searched for the parasite directly inside mosquitoes, an approach that does not require collecting blood from people. We sampled mosquitoes from two coastal districts of Odisha, India, that had already passed the required surveys and stopped treatment. We found the parasite Brugia malayi in Mansonia mosquitoes, the first time this has been shown using this molecular method in the region, although the more common filarial parasite was not detected. Our findings show that transmission can smoulder on even after an area is judged free of disease. Because India has no available rapid field test for this particular parasite, we argue that examining mosquitoes should become a routine safeguard for the countrys elimination effort.

zoology↗

bioRxiv2026

SMC Motor Proteins Operate at the Near-Minimal Forces for DNA Loop Extrusion

Loop extrusion by structural maintenance of chromosomes (SMC) complexes is essential for genome organization, yet the forces driving this process remain poorly understood. We present a coarse-grained model enabling predictive simulations of in vitro loop extrusion experiments at experimentally relevant time and length scales by matching parameters with concrete experiments. Using this model, we demonstrate that the extrusion forces generated by SMC motor proteins are just sufficient to overcome initial entropic barriers and sustain loop extrusion, highlighting that motors operate in the thermal regime. By measuring stalling tension directly, we confirm that they can be reliably determined by the Marko-Siggia equation and that varying grafting distances in experimental setups has only a marginal effect on the resulting tension. These results provide a predictive computation method for dissecting the mechanics of SMC driven genome folding.

biophysics↗

bioRxiv2025

Large-scale single-cell phylogenetic mapping of clonal evolution in the human aging esophagus

The human somatic genome evolves throughout our lifespan, producing mosaic individuals comprising clones harboring different mutations across tissues. While clonal expansions in the hematopoietic system have been extensively characterized and reported to be nearly ubiquitous, clonal mosaicism (CM) has more recently also been described across multiple solid tissues. However, outstanding questions remain about the parameters and processes of human somatic evolution in non-cancerous solid human tissues, including when clones arise, how they evolve over time, and what mechanisms lead to their expansion. Questions of timing and clonal dynamics can be addressed through phylogenetic reconstruction, which serves as a temporal microscope, while uncovering the mechanisms of expansion necessitates simultaneous phenotypic profiling. To address this gap, here we develop Single-cell Miniaturized Automated Reverse Transcription and Primary Template-directed Amplification (SMART-PTA) for joint single-cell whole-genome and whole-transcriptome sequencing for large scale and cost efficient interrogation of solid tissue CM. We established a workflow that generates hundreds of matched single-cell whole genome and transcriptome libraries within a week. We profiled phenotypically normal esophagus tissue from four aged donors and used somatic variants to build high-resolution single-cell lineages from >2,700 cells with accompanying transcriptomic information, reconstructing >70 years of somatic evolution. T cell expansions identified from T cell receptor (TCR) sequences validated the clonal structure of the single-nucleotide variant (SNV)-based phylogenies and phylogenetic cross-correlation analysis showed that epithelial cells had higher degrees of shared ancestry by spatial location compared to immune cells. Mapping mutation signatures to the phylogenetic tree revealed the emergence of tobacco/alcohol exposure-related signatures later in life, consistent with the donors exposure histories. We identified variants in driver genes that were previously reported in the phenotypically normal esophagus, detecting clonal expansions harboring mutations in genes including TP53 and FAT1. We mapped the evolution of clones with both monoallelic as well as biallelic TP53 loss, including a clone associated with high expression of cell cycling genes and higher chromosome instability. Leveraging the matched transcriptome data, we uncovered cell type biases in mutant clones, with a higher proportion of TP53 or FAT1-mutant cells in an earlier basal epithelial cell state compared to wild-type cells. We further observed copy-neutral loss of heterozygosity (CNLOH) events on chromosome 9q that spanned the NOTCH1 locus in up to [~]35% of epithelial cells. Mapping CNLOH events to the phylogenetic tree revealed a striking pattern in which CNLOH was separately acquired many times, reflecting convergent evolution. Cells with CNLOH events were biased towards the earlier basal epithelial state, suggestive of a selective advantage that leads to prevalent recurrence of chr9q CNLOH. Together, we demonstrate that SMART-PTA is an efficient, scalable approach for single-cell whole-genome and whole-transcriptome profiling to build phenotypically annotated single-cell phylogenies with enough throughput and power for application to normal tissue somatic evolution. Moreover, we reconstruct the evolutionary history of the esophageal epithelium at high scale and resolution, providing a window into the dynamics and processes that shape clonal expansions in phenotypically normal tissues throughout a lifespan.

genomics↗

bioRxiv2025

Phagocytes as Plaque Catalysts: Human Macrophages Actively Generate Pathogenic Aβ42 Fibrils with Seeding and Cross-Seeding Potency

The prevailing view frames microglia and macrophages as guardians against amyloid beta (A{beta}) accumulation in Alzheimers disease (AD). Here, we overturn this paradigm by demonstrating that human phagocytic cells--including differentiated THP-1 macrophages and iPSC-derived microglia--are not merely passive responders but active producers of extracellular, seeding-competent A{beta}42 fibrils, the amyloid species most strongly linked to parenchymal plaque formation and neurodegeneration. These cell-generated aggregates differ structurally and functionally from synthetic fibrils, exhibiting heightened seeding activity and the ability to cross-seed tau aggregation, a key driver of AD progression. Notably, A{beta}42 fibril formation in this system requires active cellular processes and is exacerbated by loss of TREM2, a major AD risk gene. Transcriptomic profiling reveals an early inflammatory response resembling microglial states observed in human AD models, positioning this system as a tractable, human-relevant platform to dissect the interplay between A{beta} aggregation, innate immunity, and genetic susceptibility. Our findings suggest that macrophages and microglia play a dual role in AD, acting both as responders and inadvertent catalysts of pathogenic amyloid formation, with implications for early therapeutic intervention. Significance StatementHow amyloid plaques emerge and spread in Alzheimers disease remains a critical unanswered question. Here, we show that human immune cells--including brain-resident microglia--can actively generate A{beta}42 fibrils, the form of amyloid most strongly linked to neurodegeneration. These cell-produced fibrils not only seed further amyloid buildup but also trigger tau aggregation, a key event in disease progression. We further demonstrate that genetic risk factors like TREM2 amplify this process. Our findings reveal a direct link between immune cell activity, genetic susceptibility, and the earliest stages of Alzheimers pathology, offering new insights into disease mechanisms and potential intervention points.

biochemistry↗

bioRxiv2025

AKNA drives neural stem cell fate transition through differential localization and coordinating the modulation of chromatin from H3K27me3 to H3K27ac

Neuronal stem cells (NSCs) play pivotal role in adult neurogenesis, however, detail mechanisms of obtaining pluripotency or undergoing differentiation remain unknown. Herein, how AT-hook protein AKNA regulates pluripotency and stemness in neuroblastoma cells is demonstrated by gene knockdown, immunofluorescence, chromatin immunoprecipitation (ChIP), localization of AKNA, signaling interactions and transcriptional activity. AKNA was abundant and mostly nuclear during induction of pluripotency and its knockdown reduced stemness, even in presence of other pluripotency factors, including OCT4 and SOX2. During the course of induction of differentiation, AKNA remain localized in the cytosol, essential for regulated differentiation. Cytosolic retention of AKNA is possibly driven by FAK signaling. In the nucleus, AKNA promotes KDM6B demethylase for H3K27me3 demethylation to H3K27, and subsequently promotes CBP/p300 to acetylate H3K27 to H3K27ac deposition on promoter region of respective genes to trigger their transcription. Data obtained from knockdown and overexpression of AKNA and KDM6B further reinforce its importance that, they physically interact to drive pluripotency and stemness. These findings establish AKNA as a critical regulator for NSCs fate determination in association with epigenetic modifiers and signaling pathway, offering potential targets for neuroblastoma therapies and regenerative medicines for neurodegenerative diseases.

molecular biology↗

bioRxiv2025

Macrophage signaling and function are regulated by distinct sterol biochemistries

Membranes require continuous reorganization of lipid components, including sterols, to dynamically alter their rigidity to deform and bend during scission events which occur during fundamental cellular functions such as endocytosis. While diseases of cholesterol biosynthesis result in reduced cellular cholesterol and accumulation of precursor sterols, limited studies have addressed the intracellular consequences of disease-associated sterol changes on the ability of eukaryotic cellular membranes to function and signal normally. Here, we utilized bone marrow-derived macrophages (BMDMs) to investigate how altered sterol content impacts macrophage signaling and membrane function. Through pharmacological inhibition of cholesterol biosynthetic enzymes, reduced cholesterol and increased levels of disease-associated sterol intermediates coincided with reduced expression of cell surface proteins and impaired macropinocytosis. Macropinocytic activity was sensitive to both reduced plasma membrane cholesterol and sterols containing functional groups substituted for the C3 hydroxyl group. Transcriptomic analyses of cholesterol-inhibited BMDMs revealed alterations in immune and chemokine signaling pathways. Decreased cholesterol was also associated with dysregulated vesicular sorting pathways and elevated expression of endosomal/lysosomal markers. Disrupted endosome expression and impaired macropinocytosis was also observed in BMDMs from mouse models of the cholesterol biosynthesis disorder Smith-Lemli-Opitz syndrome (SLOS). Our findings detail an important connection between sterol imbalance, membrane dynamics, and immune cell function.

cell biology↗

bioRxiv2025

Medin drives Aβ40 to adopt Aβ42-like fibril polymorphs in vitro

Medin, a vascular amyloid derived from MFG-E8, is the most prevalent form of localized human amyloid and co-localizes with A{beta} in Alzheimers disease and, in particular, cerebral amyloid angiopathy (CAA). While it was shown that medin can promote A{beta} aggregation, it remains unclear whether this amyloid-amyloid interaction affects the structure of the resulting fibrils. Here, we investigate how medin modulates A{beta}40 fibril assembly in vitro using cryo-electron microscopy, aggregation kinetics, and immunogold electron microscopy. We show that medin accelerates A{beta}40 aggregation, co-assembles into hybrid fibrils, and modulates fibril morphology. Cryo-EM analysis reveals two fibril populations: one corresponding to a previously described in vitro A{beta}40 morphology, and a second, previously unobserved polymorph with A{beta}42-like features, including a structured N-terminus and a compact hydrophobic C-terminal core. The presence of a peripheral, unresolved cryo-EM density near the fibril surface suggests that the new polymorph is stabilised through heterotypic interactions, yet the atomic details remain unresolved, likely due to substantial structural heterogeneity. Rather than representing a limitation, this highlights how not all determinants critical for fibril assembly are necessarily ordered or resolvable in the final fibril structure, reflecting the inherent dynamic and heterogeneous nature of amyloid interactions. Our findings provide structural evidence that heterotypic co-aggregation can redirect A{beta}40 into distinct conformational states and suggest that dynamic or transient interactions contribute to fibril polymorphism beyond what can be fully captured in static structural models.

biochemistry↗

bioRxiv2024

Complete Genome Sequence, Metabolic Profiling and Functional Studies reveal Ligilactobacillus salivarius LS-ARS2 is a Promising Biofilm-forming Probiotic with Significant Antioxidant, Antibacterial, and Antibiofilm Potential

BACKGROUNDProbiotics restore microbial balance and prevent gut-inflammation. Therefore, finding out novel probiotic strains is a demand. As gut-microbe, benefits of Ligilactobacillus salivarius (LS) are established. However, strain-specific detailed studies are limited. Here, we illustrate probiotic attributes of novel LS-ARS2 for its potential application as food-supplement and/or therapeutic to improve gut-health. METHODSWhole genome sequencing (WGS) and phylogenetic analysis confirm the strain as LS. To establish probiotic properties, acid-bile tolerance, auto-aggregation, cell-surface-hydrophobicity, biofilm-formation and adhesion-assays are performed. To ensure safety attributes, antibiotic-susceptibility, haemolytic, DNase, trypan-blue and MTT assays are done. ABTS, DPPH, superoxide, hydroxyl free radical scavenging assays are used to determine anti-oxidant potential. Antibacterial assays including co-culture assay with pathogen and pathogenic biofilm-inhibition assays are performed to explore antibacterial efficacy. To characterize metabolic-profile of LS-ARS2-derived cell-free-supernatant (CFS), HRMS analysis are carried out. Consequently, WGS-analyses predict potential molecular associations related to functional outcomes. RESULTSWe find LS-ARS2 as remarkable fast-growing strain that shows acid and bile tolerance (>60% survival rate) indicating promising gut-sustainability. High auto-aggregation capacity (>80%), robust cell-surface hydrophobicity (>85%), and adhesion efficacy to Caco-2 cells illustrate significant potential of LS-ARS2 for gut colonization. Fascinatingly, LS-ARS2 is able to form biofilm within 24 h (p<0.0001), rare among LS strains, indicating the potential of the strain for efficient stay in the gut. The strain ensures safety attributes. LS-ARS2-WGS analysis recognizes probiotic-specific determinants, predicts genomic stability, identifies orthologous-clusters for diverse functions, and predicts metabolites and bacteriocins. HRMS-studies with LS-ARS2-CFS further validate the presence of diverse beneficial metabolites with antimicrobial and immunomodulatory potential. LS-ARS2 shows significant antioxidant properties in ABTS (>60%), DPPH (>10U/mL), superoxide (>70%), and hydroxyl free radical scavenging assays (>70%). Further, LS-ARS2 shows antimicrobial activities against Gram-positive Methicillin-resistant Staphylococcus aureus (MRSA) and Gram-negative multidrug-resistant clinical strains enterotoxigenic Escherichia coli, Vibrio cholerae, and Shigella flexneri. Anti-Salmonella effect of LS-ARS2 is prominent (p<0.0001). Most interestingly, LS-ARS2-CFS inhibits MRSA-biofilm (p<0.0001), again rare among LS strains. CONCLUSIONS LS-ARS2 is a novel fast-growing, biofilm-forming probiotic with significant antioxidant, antibacterial, and anti-biofilm potentials, suggesting the promising applications of LS-ARS2 for combating pathogenic biofilms and improving gut-health. However, further in vivo studies would facilitate their potential applications.

microbiology↗

bioRxiv2024

Symmetry of loop extrusion by dimeric SMC complexes is DNA-tension-dependent

Structural maintenance of chromosome (SMC) complexes organize and regulate genomes by extruding DNA loops. During loop extrusion, DNA can be reeled into the growing loop from one or both sides, generating distinct extrusion directionality states whose physical basis remains unclear. Here, we combine single-molecule analysis and molecular dynamics simulations to investigate loop extrusion directionality across SMC complexes. We show that dimeric Smc5/6 and Wadjet predominantly perform two-sided loop extrusion during initial loop growth, whereas monomeric condensin exhibits one-sided extrusion, consistent with a relationship between stoichiometry and extrusion directionality. Surprisingly, however, cohesin predominantly exhibits one-sided extrusion despite functioning as a dimeric complex. Notably, dimeric Smc5/6 and Wadjet progressively transition from two-sided to one-sided extrusion as loop growth matures. Simulations and force-dependent analysis reveal that loop extrusion directionality is governed by a tension-dependent transition in dimeric motors. Increasing DNA tension erodes two-sided extrusion near the motor stalling force, driving a tug-of-war regime in which competing motors transiently dominate one another. This transition promotes one-sided extrusion during mature Smc5/6 and Wadjet-mediated loop extrusion and places cohesin, which has a comparatively low stalling force ([~]0.1 pN), constitutively near this regime. Together, our findings establish loop extrusion directionality as a dynamic emergent property governed by SMC stoichiometry, DNA tension, and stochastic motor competition.

biophysics↗

bioRxiv2024

Mannose receptor (MRC1) mediates uptake of dextran in macrophages via receptor-mediated endocytosis

Macrophages maintain surveillance of their environment using receptor-mediated endocytosis and pinocytosis. Receptor-mediated endocytosis allows macrophages to recognize and internalize specific ligands whereas macropinocytosis non-selectively internalizes extracellular fluids and solutes. Here, CRISPR/Cas9 whole-genome screens were used to identify genes regulating constitutive and growth factor-stimulated dextran uptake in murine bone-marrow derived macrophages (BMDM). The endocytic mannose receptor c-type 1 (Mrc1, also known as CD206) was a top hit in the screen. Targeted gene disruptions of Mrc1 reduced dextran uptake but had little effect on uptake of Lucifer yellow, a fluid-phase marker. Other screen hits also differentially affected the uptake of dextran and Lucifer yellow, indicating the solutes are internalized by different mechanisms. We further deduced that BMDMs take up dextran via MRC1-mediated endocytosis by showing that competition with mannan, a ligand of MRC1, as well as treatment with Dyngo-4a, a dynamin inhibitor, reduced dextran uptake. Finally, we observed that IL4-treated BMDM internalize more dextran than untreated BMDM by upregulating MRC1 expression. These results demonstrate that dextran is not an effective marker for the bulk uptake of fluids and solutes by macropinocytosis since it is internalized by both macropinocytosis and receptor-mediated endocytosis in cells expressing MRC1. This report identifies numerous genes that regulate dextran internalization in primary murine macrophages and predicts cellular pathways and processes regulating MRC1. This work lays the groundwork for identifying specific genes and regulatory networks that regulate MRC1 expression and MRC1-mediated endocytosis in macrophages. Significance StatementO_LIMacrophages constantly survey and clear tissues by specifically and non-specifically internalizing debris and solutes. However, the molecular mechanisms and modes of regulation of these endocytic and macropinocytic processes are not well understood. Here, CRISPR/Cas9 whole genome screens were used to identify genes regulating uptake of dextran, a sugar polymer that is frequently used as a marker macropinocytosis, and compared with Lucifer yellow, a fluorescent dye with no known receptors. C_LIO_LIThe authors identified the mannose receptor as well as other proteins regulating expression of the mannose receptor as top hits in the screen. Targeted disruption of Mrc1, the gene that encodes mannose receptor, greatly diminished dextran uptake but had no effect on cellular uptake of Lucifer yellow. Furthermore, exposure to the cytokine IL4 upregulated mannose receptor expression on the cell surface and increased uptake of dextran with little effect on Lucifer yellow uptake. Studies seeking to understand regulation of macropinocytosis in macrophages will be confounded by the use of dextran as a fluid-phase marker. C_LIO_LIMRC1 is a marker of alternatively activated/anti-inflammatory macrophages and is a potential target for delivery of therapeutics to macrophages. This work provides the basis for mechanistic underpinning of how MRC1 contributes to the receptor-mediated uptake of carbohydrates and glycoproteins from the tissue milieu and distinguishes genes regulating receptor-mediated endocytosis from those regulating the bona fide fluid-phase uptake of fluids and solutes by macropinocytosis. C_LI

cell biology↗

bioRxiv2024

SH3BP2 regulates the organization of the neuromuscular synapses through protein-driven phase separation

The molecular mechanisms underlying the development and maintenance of the neuromuscular junction are poorly understood, even though the malfunction of these specialized synapses is associated with severe genetic and autoimmune disorders. The identity of factors controlling the maintenance mechanisms of postsynaptic acetylcholine receptors (AChR) in high-density has been elusive and is of great interest to the pharma industry, searching for possible new targets for disease interventions. Here, we report the identification of a scaffold protein SH3BP2, which exhibits polyvalent interaction with the dystrophin-glycoprotein complex (DGC) and AChR pentamers, promoting AChR clustering through phase separation. Muscle-specific SH3BP2 deletion in mice leads to impaired organization of the neuromuscular synapses, defects in synaptic transmission, and reduced muscle strength. Our studies identified a novel regulator of the postsynaptic machinery involved in clustering AChR and linking it to the DGC.

neuroscience↗

bioRxiv2024

Loop extrusion-mediated plasmid DNA cleavage by the bacterial SMC Wadjet complex

Structural maintenance of chromosomes (SMC) protein complexes play pivotal roles in genome organization and maintenance across all domains of life. In prokaryotes, SMC family Wadjet complexes structurally resemble the widespread Muk-BEF genome-organizing complexes but serve a defensive role by inhibiting plasmid transformation. We previously showed that Wadjet specifically cleaves circular DNA; however, the molecular mechanism underlying DNA substrate recognition remains unclear. Here, we use in vitro single-molecule imaging to directly visualize DNA loop extrusion and plasmid cleavage by Wadjet. We find that Wadjet is a symmetric DNA loop extruder that simultaneously reels in DNA from both sides of a growing loop and that this activity requires a dimeric JetABC supercomplex containing two dimers of the JetC motor subunit. On surface-anchored plasmid DNAs, Wadjet extrudes the full length of a 44 kilobase pair plasmid, stalls, and then cleaves DNA. Our findings reveal the role of loop extrusion in the specific recognition and elimination of plasmids by Wadjet, and establish loop extrusion as an evolutionarily conserved mechanism among SMC complexes across kingdoms of life.

molecular biology↗