Biophysics Seminar- “In vivo Single Particle Tracking in Diffraction-sized Biomolecular Condensates” with Alisha Budhathoki (Spille Lab, UIC)
Biophysics Seminar
January 31, 2025
2:00 PM - 3:00 PM
Location
SES 2214
Calendar
Download iCal FileAlisha Budhathoki
Spille Lab, Department of Physics
Spille Lab, Department of Physics
Biomolecular condensates are implicated in a wide range of biological processes. Transcription condensates concentrate important regulatory factors at super-enhancers that control the
expression of cell type-specific genes. They could explain how enhancers can facilitate the expression of target genes over long distances in sequence space. The small size of transcription
condensates at or below the diffraction limit (300 nm) makes it difficult to probe their material properties and test the phase separation model for their formation. Here, we use single particle
tracking to examine the dynamics of individual RNA Polymerase II molecules inside transcription condensates in live mouse embryonic stem cells. We compare their mobility within
condensates and in the nucleoplasm. Our results reveal a DNA-bound population and two distinct mobile states. Surprisingly, we find that inside transcription condensates virtually all Pol
II molecules are in the immobile state. This finding is corroborated by our observation that Pol II in transcription condensates is highly serine 5 phosphorylated. Our results suggest that Pol II
in transcription condensates is mostly initiating and bound to DNA near the promoter. Inhibition of transcription initiation increases Pol II mobility inside the nucleus and at condensates.
Notably, the Mediator fraction of condensates persists under initiation inhibition while Pol II is gradually purged from condensates. We hypothesize that efficient loading of Pol II onto DNA
drives the increased local Pol II concentration inside condensates. Our results demonstrate that single particle tracking presents a valuable tool for examining how transcription regulatory
proteins experience the condensate environment and will help us develop biophysical models for their mechanistic role in transcription regulation.
expression of cell type-specific genes. They could explain how enhancers can facilitate the expression of target genes over long distances in sequence space. The small size of transcription
condensates at or below the diffraction limit (300 nm) makes it difficult to probe their material properties and test the phase separation model for their formation. Here, we use single particle
tracking to examine the dynamics of individual RNA Polymerase II molecules inside transcription condensates in live mouse embryonic stem cells. We compare their mobility within
condensates and in the nucleoplasm. Our results reveal a DNA-bound population and two distinct mobile states. Surprisingly, we find that inside transcription condensates virtually all Pol
II molecules are in the immobile state. This finding is corroborated by our observation that Pol II in transcription condensates is highly serine 5 phosphorylated. Our results suggest that Pol II
in transcription condensates is mostly initiating and bound to DNA near the promoter. Inhibition of transcription initiation increases Pol II mobility inside the nucleus and at condensates.
Notably, the Mediator fraction of condensates persists under initiation inhibition while Pol II is gradually purged from condensates. We hypothesize that efficient loading of Pol II onto DNA
drives the increased local Pol II concentration inside condensates. Our results demonstrate that single particle tracking presents a valuable tool for examining how transcription regulatory
proteins experience the condensate environment and will help us develop biophysical models for their mechanistic role in transcription regulation.
Date posted
Jan 15, 2025
Date updated
Feb 4, 2025