Biology subjects

Budhathoki, A.

Publications and source records attributed to Budhathoki, A..

4 recordsLinked to original sources

bioRxiv2026

Extracting anomalous diffusion parameters from multi-state ensembles of short single molecule trajectories.

Single-molecule tracking measures the stochastic motion of individual biomolecules in the cellular environment. Statistical analysis of trajectory ensembles is required to gain insight into the biophysical nature of mobility states and molecular interactions that they reflect. Mobility states can be parameterized by a generalized diffusion coefficient and anomalous exponent. Experimental constraints such as finite track length and localization precision limit how accurately these parameters can be determined. We compare the performance of analysis methods to recover the input parameters from ensembles of simulated single molecule tracks from different states spanning the range of anomalous diffusive behaviors observed in the cell nucleus. We further develop a framework to quantify error rates in the assignment of mobility states to individual molecules based on recall rates and precision. Our analysis shows that single-track analysis methods are superior to bulk methods in their ability to recover parametric descriptors from mixed populations. The most complete description is obtained by combining outputs from different tools. Our work provides a guide to assess the accuracy of analyses and obtain the most accurate parametric description of experimental single particle tracking data. Statement of significanceExperimental single particle tracking data provides rich insight into molecular interactions directly in living cells. But data analysis depends critically on choosing the correct diffusion model and appropriate tools to extract accurate information. Importantly, it is usually not obvious from the output of a method whether the results are accurate or not. In this work, we use ensembles of tracks simulated with fractional Brownian motion methods to characterize the impact of track length and localization precision on analysis outcomes. We elaborate on specific strengths and weaknesses of commonly used and newly developed analysis tools to provide a template for thorough assessment and quantification of error rates in experimental data analysis.

biophysics↗

bioRxiv2026

IDRs lead the way: Cooperativity between intrinsically disordered regions and structured interaction domains drives enrichment in transcription condensates

Many biomolecular condensates are thought to form through phase separation driven by weak and multivalent, non-stoichiometric interactions between intrinsically disordered protein regions (IDRs). IDRs are abundant in the transcription-related proteome. In vitro, different transcription-related IDRs coalesce into the same droplets, providing support for this IDR-centric paradigm of protein enrichment in transcription condensates in the cell nucleus. But our experiments show that IDRs are not sufficient to account for the degree of enrichment observed for full-length proteins in endogenous transcription condensates. Instead, we find a pattern in which IDRs facilitate engagement of structured interaction domains with a binding substrate. Instead, we find a pattern in which IDRs facilitate engagement of structured interaction domains with a binding substrate. Our results indicate that the role of IDRs in transcription condensates requires further investigation with tools that assess their mode of action in situ. Understanding the role of different protein domains and their interplay will also be important for interpreting biotechnological assays that utilize parts of condensate forming proteins.

cell biology↗

bioRxiv2026

Transcription condensates are promoter hubs that enhance transcriptional bursts

Transcription of eukaryotic genes by RNA Polymerase II occurs in temporal bursts and spatial clusters. It is regulated by dozens of transcription factor and coactivator proteins and guided by epigenetic histone marks. Colocalization of transcription machinery in dense foci suggests that cooperative effects orchestrate the process. Factory or condensate models provide a framework for the spatial assembly of the transcription machinery at highly active chromatin loci. But conventional methods lack the resolution to determine how chromatin regulatory elements interact with spatial clusters of the transcription machinery, and whether chromatin structural features modulate functional output. Here, we use super-resolution microscopy to elucidate nanoscale organization of regulatory chromatin at Pol II clusters across scales. We find that Pol II clusters exist on a continuous spectrum of sizes and represent promoter chromatin hubs. We uncover a layered organization of regulatory chromatin, where Pol II clusters form at H3K27ac and H3K4me3-rich domains while H3K4me1 positions peripherally at the surface of large Pol II clusters. Perturbation experiments are consistent with a model in which cohesin loop extrusion forms the active chromatin scaffold underlying transcription assemblies while condensate-driven interactions play only a minor role in genome organization at these sites. Importantly, the number and size of transcriptional burst size increases with Pol II cluster size, revealing directly the cooperative benefits of transcription organization in promoter hubs and a functional consequence of local chromatin structure.

cell biology↗

bioRxiv2026

Large RNA polymerase II condensates are promoter-centric assemblies associated with early stages of transcription at all expressed genes

Transcriptional condensates concentrate the machinery required for RNA polymerase II mediated transcription. These structures range from numerous small, short-lived species, to a handful of larger, stable assemblages. Large condensates have been implicated in driving potent transcription of several super-enhancer regulated genes, yet the underlying mechanisms and the range of their client genes remain unclear. Here, we developed a biochemical approach which combines density gradient centrifugation and affinity purification to partially purify large transcriptional condensates from nuclei, allowing systematic characterization of their nucleic acid components. We find that transcriptional condensate isolates engage thousands of gene promoters and harbor the nascent transcriptome, but do not stably co-purify with distal enhancers. Binding patterns of RNA polymerase II within condensates suggest these structures could facilitate promoter escape and promoter-proximal pause release. Together, our work supports a promoter-centric condensate organization and paves the way towards understanding the functional link between condensate architecture and nascent transcription.

molecular biology↗