Biology subjects

Wijeratne, S.

Publications and source records attributed to Wijeratne, S..

12 recordsLinked to original sources

bioRxiv2026

Resolving Orsay Virus δ Protein Architecture Using Molecular Rulers in Single-Molecule Force Spectroscopy

Understanding the mechanical stability and architecture of viral proteins can provide valuable information about their biological function, but it remains a significant biophysical challenge. This study employs single-molecule force spectroscopy (SMFS) to investigate the multi-domain architecture of the Orsay virus {delta} protein, which lacks repeat structures and exhibits weak unfolding peaks. We engineered a construct using titin (I27)4 domains as an internal molecular ruler, enabling us to bracket the {delta} protein peaks to determine domain length and identify unfolding forces with an atomic force microscope (AFM). To address limitations of one-dimensional (1D) force distributions in resolving overlapping structural states, we created a two-dimensional (2D) mechano-structural signature map. By plotting kinetic stability (unfolding force F) against physical structural footprint (domain length L), we distinguished distinct unfolding domains, successfully separating degenerate 1D data into two statistically distinct populations corresponding to the {delta} proteins internal domain (I) and C-terminal domain (C). This label-free method provides the first mechanical evidence of the {delta} proteins multi-domain architecture. It establishes a robust, multi-dimensional framework for decoding the mechanics of complex biomolecular assemblies in their native state.

biophysics↗

bioRxiv2025

Multiomic characterization, early detection, and therapeutic targeting of myeloid sarcoma

Myeloid sarcoma, an aggressive extramedullary subtype of acute myeloid leukemia (AML), occurs in [~]10% of patients, and has not yet been included in large-scale genomic studies. The critical biological changes that drive tumor evolution are unknown, its detection in asymptomatic patients remains a clinical challenge, and treatment options are limited as patients are often excluded from clinical trials, rendering it a neglected disease entity. Based on comprehensive multi-omic profiling, we demonstrate that myeloid sarcoma evolves from medullary AML with distinct sitespecific clonal evolution patterns. Additionally, we show that circulating tumor DNA sequencing can serve as a non-invasive method for molecular profiling of myeloid sarcoma, offering a novel avenue in molecular diagnostics. We characterize unique transcriptional profiles of myeloid sarcoma, reflecting immune evasion and adaptation to an extramedullary microenvironment. We provide evidence for a key role of RAS pathway activation and demonstrate in murine models of myeloid sarcoma that RAS inhibition effectively reduces tumor burden. Overall, our data highlight key differences between medullary AML and myeloid sarcoma including universal molecular evolution and RAS pathway activation as hallmarks of the disease and nominate RAS inhibition as a promising therapeutic strategy for patients with myeloid sarcoma.

cancer biology↗

bioRxiv2025

A minimum module for positioning the Chromosomal Passenger Complex at the cell center for cytokinesis

The Aurora B kinase-containing Chromosomal Passenger Complex (CPC) is an essential regulator of cytokinesis, the final and irreversible step of cell division. During anaphase, the CPC concentrates at the spindle midzone - a bundle of overlapping antiparallel microtubules organized at the cell center. How CPC is selectively enriched at the midzone within the dense, heterogeneous, and dynamic spindle microtubule network is unknown. Here, we define a minimal CPC midzone enrichment module. We show that the maximal enrichment of CPC at antiparallel microtubule overlaps requires PRC1-crosslinked microtubules and the interaction of CPC with two mitotic kinesins, KIF4A and KIF20A. We find that the two motors exhibit a division of labor: KIF20A delivers CPC from non-overlapping microtubules, and KIF4A retains CPC at PRC1-crosslinked overlaps. Conditional depletion of KIF4A in mitotic cells reveals that it is required for CPC localization at the spindle midzone in anaphase. Taken together, our findings reveal how the collective activity of two kinesins enables navigation through the complex microtubule network of the spindle to organize kinase signaling at the cell center for cytokinesis.

cell biology↗

bioRxiv2025

Micron-scale protein transport along microtubules by kinesin-driven shepherding

How microscopic interactions give rise to cellular-scale order in the crowded environment of cells remains an open problem. Far-from-equilibrium mixtures of active and passive molecules self-organize, but the principles remain unclear. Microtubules are cytoskeletal polymers composed of 13 protofilaments bound by mixtures of actively moving motors and passively diffusing microtubule-associated proteins (MAPs), serving as a model system for self-organization on a multi-lane lattice. Here, we combine computational modeling, analytic theory, and in vitro reconstitution to demonstrate that motors can "shepherd" diffusive MAPs by rectifying Brownian motion into net directional drift without motor-MAP binding. Our model reveals that biased movement increases when fewer protofilaments are accessible or when lateral diffusion between protofilaments is limited, identifying local dimensionality and diffusion as key parameters governing spatial patterning. Notably, shepherding depends not on individual motor processivity but on the total number of motors bound to microtubules. We experimentally recapitulate micron-scale shepherding with a kinesin-1 motor (K401) and a diffusive MAP (PRC1), which show no detectable binding. These findings reveal shepherding as an emergent mechanism by which ensembles of motors generate micron-scale spatial patterning and transport of MAPs without direct binding interactions.

biophysics↗

bioRxiv2023

Unveiling the Immune Dynamics of Neisseria Persistent Oral Colonization: A Roadmap for Innovative Vaccine Strategies

Commensal bacteria are crucial in maintaining host physiological homeostasis, immune system development, and protection against pathogens. Despite their significance, the factors influencing persistent bacterial colonization and their impact on the host still need to be fully understood. Animal models have served as valuable tools to investigate these interactions, but most have limitations. The bacterial genus Neisseria, which includes both commensal and pathogenic species, has been studied from a pathogenicity to humans perspective, but lacks models that study immune responses in the context of long-term persistence. Neisseria musculi, a recently described natural commensal of mice, offers a unique opportunity to study long-term host-commensal interactions. In this study, for the first time we have used this model to study the transcriptional, phenotypic, and functional dynamics of immune cell signatures in the mucosal and systemic tissue of mice in response to Neisseria musculi colonization. We found key genes and pathways vital for immune homeostasis in palate tissue, validated by flow cytometry of immune cells from lung, blood and spleen. This study offers a novel avenue for advancing our understanding of host-bacteria dynamics and may provide a platform for developing efficacious interventions against mucosal persistence by pathogenic Neisseria. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=151 SRC="FIGDIR/small/572139v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@1728fdaorg.highwire.dtl.DTLVardef@426a5forg.highwire.dtl.DTLVardef@55a90forg.highwire.dtl.DTLVardef@1ce3ff6_HPS_FORMAT_FIGEXP M_FIG C_FIG

immunology↗

bioRxiv2022

MUCOSECRETORY LUNG DISEASE: DIFFERENT ASSEMBLIES OF JAG1 AND JAG2 DETERMINE TRACHEOBRONCHIAL CELL FATE

Mucosecretory lung disease compromises airway epithelial function and is characterized by goblet cell hyperplasia and ciliated cell hypoplasia. These cell types are derived from tracheobronchial stem/progenitor cells via a Notch dependent mechanism. Although specific arrays of Notch receptors regulate cell fate determination, the function of the ligands Jagged1 (JAG1) and Jagged2 (JAG2) is unclear. This study used primary human bronchial air-liquid- interface cultures, gamma secretase inhibition, and neutralizing antibodies to show: 1) JAG1 and JAG2 were necessary for secretory progenitor cell fate determination; 2) JAG2 suppressed squamous differentiation; and 3) pausing of the ciliated cell differentiation process after Notch inhibition. Histological, cell fractionation, cell surface biotinylation, and ubiquitination analyses demonstrated that all cells were JAG1 positive but that little JAG1 was present on the cell surface. In contrast, JAG2 was expressed in a positive-negative pattern and was abundant on the cell surface. Glycogen synthase kinase 3 (GSK3) and tankyrase inhibition studies showed that GSK3 regulated JAG2 trafficking, and that this mechanism was WNT-independent. Collectively, these data indicate that variation in JAG2 trafficking creates regions of high, medium, and low ligand expression. Thus, distinct assemblies of JAG1 and JAG2 may regulate Notch signal strength and determine the fate of tracheobronchial stem/progenitor cells. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=86 SRC="FIGDIR/small/478334v2_ufig1.gif" ALT="Figure 1"> View larger version (15K): org.highwire.dtl.DTLVardef@37bb51org.highwire.dtl.DTLVardef@eaeaf5org.highwire.dtl.DTLVardef@e791dcorg.highwire.dtl.DTLVardef@1193d73_HPS_FORMAT_FIGEXP M_FIG O_FLOATNOGraphical AbstractC_FLOATNO Different assemblies of JAG1 and JAG2 may determine Notch signal strength and cell fate within the tracheobronchial epithelium. A cell which interacts with JAG1+ cells (blue squares) receives a low Notch signal (light yellow square). A cell which interacts with a mixture of JAG1+ and JAG1+/JAG2+ cells (purple squares) receives a medium (med) Notch signal (medium yellow square). A cell which interacts with JAG1+/JAG2+ cells receives a high Notch signal (bright yellow square). C_FIG

cell biology↗

bioRxiv2021

Discovery of Clinically Relevant Fusions in Pediatric Cancer

AO_SCPLOWBSTRACTC_SCPLOWO_ST_ABSBackgroundC_ST_ABSPediatric cancers typically have a distinct genomic landscape when compared to adult cancers and frequently carry somatic gene fusion events that alter gene expression and drive tumorigenesis. Sensitive and specific detection of gene fusions through the analysis of next-generation-based RNA sequencing (RNA-Seq) data is computationally challenging and may be confounded by low tumor cellularity or underlying genomic complexity. Furthermore, numerous computational tools are available to identify fusions from supporting RNA-Seq reads, yet each algorithm demonstrates unique variability in sensitivity and precision, and no clearly superior approach currently exists. To overcome these challenges, we have developed an ensemble fusion calling approach to increase the accuracy of identifying fusions. ResultsOur ensemble fusion detection approach utilizes seven fusion calling algorithms: Arriba, CICERO, FusionMap, FusionCatcher, JAFFA, MapSplice, and STAR-Fusion, which are packaged as a fully automated pipeline using Docker and AWS serverless technology. This method uses paired end RNA-Seq sequence reads as input, and the output from each algorithm is examined to identify fusions detected by a consensus of at least three algorithms. These consensus fusion results are filtered by comparison to an internal database to remove likely artifactual fusions occurring at high frequencies in our internal cohort, while a "known fusion list" prevents failure to report known pathogenic events. We have employed the ensemble fusion-calling pipeline on RNA-Seq data from 229 patients with pediatric cancer or blood disorders studied under an IRB-approved protocol. The samples consist of 138 central nervous system tumors, 73 solid tumors, and 18 hematologic malignancies or disorders. The combination of an ensemble fusion-calling pipeline and a knowledge-based filtering strategy identified 67 clinically relevant fusions among our cohort (diagnostic yield of 29.3%), including RBPMS-MET, BCAN-NTRK1, and TRIM22-BRAF fusions. Following clinical confirmation and reporting in the patients medical record, both known and novel fusions provided medically meaningful information. ConclusionsOur ensemble fusion detection pipeline offers a streamlined approach to discover fusions in cancer, at higher levels of sensitivity and accuracy than single algorithm methods. Furthermore, this method accurately identifies driver fusions in pediatric cancer, providing clinical impact by contributing evidence to diagnosis and, when appropriate, indicating targeted therapies.

genomics↗

bioRxiv2021

REPEATED INJURY PROMOTES TRACHEOBRONCHIAL TISSUE STEM CELL ATTRITION

Chronic lung disease has been attributed to stem cell aging and/or exhaustion. To address this issue, we investigated the lifespan of tracheobronchial tissue stem cells (TSC) over time and in response to repeated injury. Chromatin and nucleotide labeling studies in mice indicated that: 1) injury activated a subset of the TSC pool and that this process conserved TSC over time; and 2) activated TSC were predisposed to further proliferation and this activated state lead to terminal differentiation. Analysis of human TSC and clonal isolates indicated that repeated TSC proliferation led to telomere shortening and analysis of TSC from Dyskeratosis Congenita donors indicated that mutations in telomere biology genes accelerated TSC depletion. RNAseq and functional studies indicated that human TSC terminated as a secretory committed cell. These data support a model in which a repeated epithelial injury depletes the TSC pool and initiates the abnormal repair associated with chronic lung disease.

cell biology↗

bioRxiv2020

Motor guidance by long-range communication through the microtubule highway

Coupling of motor proteins within arrays drives muscle contraction, flagellar beating, chromosome segregation, and other biological processes. Current models of motor coupling invoke either direct mechanical linkage or protein crowding, which rely on short-range motor-motor interactions. In contrast, coupling mechanisms that act at longer length scales remain largely unexplored. Here we report that microtubules can physically couple motor movement in the absence of short-range interactions. The human kinesin-4 Kif4A changes the run-length and velocity of other motors on the same microtubule in the dilute binding limit, when 10-nm-sized motors are separated by microns. This effect does not depend on specific motor-motor interactions because similar changes in Kif4A motility are induced by kinesin-1 motors. A micron-scale attractive interaction potential between motors is sufficient to recreate the experimental results in a computational model. Unexpectedly, our theory suggests that long-range microtubule-mediated coupling not only affects binding kinetics but also motor mechanochemistry. Therefore, motors can sense and respond to motors bound several microns away on a microtubule. These results suggest a paradigm in which the microtubule lattice, rather than being merely a passive track, is a dynamic medium responsive to binding proteins to enable new forms of collective motor behavior.

biophysics↗

bioRxiv2019

Leveraging targeted sequencing for non-model species: a step-by-step guide to obtain a reduced SNP set and a pipeline to automate data processing in the Antarctic Midge, Belgica antarctica.

The sequencing of whole or partial (e.g. reduced representation) genomes are commonly employed in molecular ecology and conservation genetics studies. However, due to sequencing costs, a trade-off between the number of samples and genome coverage can hinder research for non-model organisms. Furthermore, the processing of raw sequences requires familiarity with coding and bioinformatic tools that are not always available. Here, we present a guide for isolating a set of short, SNP-containing genomic regions for use with targeted amplicon sequencing protocols. We also present a python pipeline--PypeAmplicon-- that facilitates processing of reads to individual genotypes. We demonstrate the applicability of our method by generating an informative set of amplicons for genotyping of the Antarctic midge, Belgica antarctica, an endemic dipteran species of the Antarctic Peninsula. Our pipeline analyzed raw sequences produced by a combination of high-multiplexed PCR and next-generation sequencing. A total of 38 out of 47 (81%) amplicons designed by our panel were recovered, allowing successful genotyping of 42 out of 55 (76%) targeted SNPs. The sequencing of [~]150 bp around the targeted SNPs also uncovered 80 new SNPs, which complemented our analyses. By comparing overall patterns of genetic diversity and population structure of amplicon data with the low-coverage, whole-genome re-sequencing (lcWGR) data used to isolate the informative amplicons, we were able to demonstrate that amplicon sequencing produces information and results similar to that of lcWGR. Our methods will benefit other research programs where rapid development of population genetic data is needed but yet prevented due to high expense and a lack of bioinformatic experience.

evolutionary biology↗

bioRxiv2019

Ribosomal stalk proteins RPLP1 and RPLP2 promote biogenesis of flaviviral and cellular multi-pass transmembrane proteins

Dengue virus (DENV) and other mosquito-borne flaviviruses are highly dependent on the ribosomal stalk proteins, RPLP1 and RPLP2 (RPLP1/2), for efficient infection. Here, we show that RPLP1/2 function to relieve ribosome pausing within the DENV envelope coding sequence, leading to enhanced protein stability. We used ribosome profiling to evaluate viral and cellular translation in RPLP1/2-depleted cells. This revealed that ribosomes pause in the sequence coding for the N-terminus of the envelope protein, immediately downstream of sequences encoding two adjacent transmembrane domains (TMDs). RPLP1/2 function to enhance ribosome elongation at this position and increase viral protein stability, possibly by improving co-translational folding of DENV proteins. We also analyzed the effects of RPLP1/2 depletion on cellular translation. We find that RPLP1/2 affects ribosome density for a small subset of cellular mRNAs. However, meta-analysis of ribosome positions on all cellular mRNAs revealed slightly increased accumulation of ribosomes downstream of start codons in RPLP1/2-depleted cells, suggesting that RPLP1/2 enhance elongation efficiency. Importantly, we found that ribosome density on mRNAs encoding multiple TMDs was disproportionately affected by RPLP1/2 knockdown, implying a role for RPLP1/2 in transmembrane protein biogenesis. Together, our findings reveal insights into the function of RPLP1/2 in DENV and cellular translation.

molecular biology↗

bioRxiv2017

Geometry of antiparallel microtubule bundles regulates relative sliding and stalling by PRC1 and Kif4A

Motor and non-motor crosslinking proteins play critical roles in determining the size and stability of microtubule-based architectures. Currently, we have a limited understanding of how geometrical properties of microtubule arrays, in turn, regulate the output of crosslinking proteins. Here we investigate this problem in the context of microtubule sliding by two interacting proteins: the non-motor crosslinker PRC1 and the kinesin Kif4A. The collective activity of PRC1 and Kif4A also results in their accumulation at microtubule plus-ends ( end-tag). Sliding stalls when the end-tags on antiparallel microtubules collide, forming a stable overlap. Interestingly, we find that structural properties of the initial array regulate PRC1-Kif4A mediated microtubule organization. First, sliding velocity scales with initial microtubule-overlap length. Second, the width of the final overlap scales with microtubule lengths. Our analyses reveal how micron-scale geometrical features of antiparallel microtubules can regulate the activity of nanometersized proteins to define the structure and mechanics of microtubule-based architectures.

biophysics↗