Steen, S. B. | bioRxiv Science

bioRxiv2026

Substrate recognition, not sequestration, drives the engagement of an H3K9 methyltransferase in living cells

The histone H3K9 methyltransferase Clr4 is essential for heterochromatin formation in Schizosaccharomyces pombe, yet how it searches for and engages chromatin in vivo remains unclear. Using live-cell single-molecule tracking of PAmCherry-Clr4, we quantified how perturbations to Clr4 alter its diffusion, search behavior, and residence times. Chromodomain and SET-domain Clr4 mutants move faster and more isotropically and have reduced residence times at heterochromatin, reflecting impaired substrate recognition. In contrast, deleting Swi6, which has been proposed to sequester Clr4, does not reduce the slow-state fraction or alter Clr4 diffusion, indicating that chromatin engagement is intrinsic to Clr4 rather than HP1-dependent. Anisotropy analysis at short and intermediate displacements indicates that Clr4 does not explore chromatin by simple three-dimensional diffusion but through a guided, distance-dependent search in which it repeatedly samples nearby nucleosomes before disengaging. Across all other perturbations we examined, such as deletion of the CLRC component Rik1, impaired Clr4 ubiquitination, and using cells with an unmethylatable H3K9R substrate, Clr4 dynamics were only modestly affected, and a chromatin-associated population persisted in every background. This robustness indicates that the chromodomain and SET domains are the primary determinants of how Clr4 engages chromatin in vivo, allowing it to continuously sample the genome while maintaining a stable bound population. Our results suggest how the promiscuous sampling of chromatin may also enable Clr4 to establish novel sites of heterochromatin during adaptation.

biophysics↗

bioRxiv2024

The Endoplasmic Reticulum as an Active Liquid Network

The peripheral endoplasmic reticulum (ER) forms a dense, interconnected, and constantly evolving network of membrane-bound tubules in eukaryotic cells. While individual structural elements and the morphogens that stabilize them have been described, a quantitative understanding of the dynamic large-scale network topology remains elusive. We develop a physical model of the ER as an active liquid network, governed by a balance of tension-driven shrinking and new tubule growth. This minimalist model gives rise to steady-state network structures with density and rearrangement timescales predicted from the junction mobility and tubule spawning rate. Several parameter-independent geometric features of the liquid network model are shown to be representative of ER architecture in live mammalian cells. The liquid network model connects the time-scales of distinct dynamic features such as ring closure and new tubule growth in the ER. Furthermore, it demonstrates how the steady-state network morphology on a cellular scale arises from the balance of microscopic dynamic rearrangements. SIGNIFICANCEThe peripheral endoplasmic reticulum (ER) forms a continuous, dynamic network of tubules that plays an important role in protein sorting, export, and quality control, as well as cellular signaling and stress response. Elucidating how the unique morphology of the ER arises and supports its function is critical to developing a mechanistic understanding of the many neurological diseases associated with ER structural perturbations. We develop a physical model of the ER as an active liquid network to understand how its cellular-scale structure emerges from small-scale dynamic rearrangements. The model demon-strates how key features of ER architecture can arise from a balance of tubule growth and tension-driven sliding. This work provides insight into the fundamental physical mechanisms underlying the emergent morphology of the ER.

biophysics↗