Biology subjects

Payne-Dwyer, A.

Publications and source records attributed to Payne-Dwyer, A..

4 recordsLinked to original sources

bioRxiv2026

DNA gyrase in live bacteria forms liquid condensates through weak multivalent bonding of excess GyrB

Type IIA bacterial topoisomerase DNA gyrase, a GyrA/GyrB heterotetramer, has crucial roles maintaining transcription and DNA replication by relaxing positive DNA supercoils through introducing negative supercoils. However, rates of gyrase-catalysed supercoiling in vitro cannot explain much higher rates required in vivo. To address this puzzle, we used high-speed single-molecule fluorescence imaging of GyrA/GyrB reporters in live Escherichia coli, indicating that cells contain [~]40% more GyrB than GyrA expressed in a diffuse pool or in clusters whose mobility depends on whether they are bound to DNA. Unexpectedly, we discovered that clusters are non-stoichiometric containing [~]150% more GyrB than GyrA, significantly greater than the cellular average, with fluorescence recovery after photobleaching revealing that clustered GyrA and GyrB behave as a liquid whose abundance can be increased by applying gyrase-targeting antibiotics. Structural docking indicates that the liquid state is stabilised through excess GyrB progressively binding to existing clusters via weak, multivalent interactions. By operating in liquid condensates, A2B2 that dissociates from DNA can rebind rapidly instead of diffusing away, increasing enzyme processivity to enable multiple rounds of catalysis that can keep pace with transcription and DNA replication in vivo. This demonstrates a new role for condensates of overcoming kinetic limitations imposed by diffusion. Graphical abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=158 SRC="FIGDIR/small/744598v1_ufig1.gif" ALT="Figure 1"> View larger version (53K): org.highwire.dtl.DTLVardef@1514d4aorg.highwire.dtl.DTLVardef@a95ceeorg.highwire.dtl.DTLVardef@11417daorg.highwire.dtl.DTLVardef@2a2856_HPS_FORMAT_FIGEXP M_FIG C_FIG

biophysics↗

bioRxiv2026

Sticker number modulates pyrenoid condensate assembly to support algal fitness

The valency of intrinsically disordered proteins underpins liquid-liquid phase separation (LLPS), yet how this parameter shapes condensate function and cellular fitness remains poorly understood. Here we exploit the algal pyrenoid-a minimal, two component LLPS system-to directly link condensate properties to physiological performance. Pyrenoid assembly is driven by a disordered, multivalent Linker protein that binds Rubisco at symmetry-related surface sites, with the number of binding motifs ("stickers") varying across species. Using Chlamydomonas reinhardtii, we systematically tuned sticker number from two to nine and examined effects on Rubisco condensation, pyrenoid architecture and CO2 fixation. Three stickers were sufficient for condensation in vitro, but at least four were required for pyrenoid assembly in vivo. Cryo-electron tomography and single-molecule tracking revealed that increasing sticker number enhances Rubisco packing and mobility, while time-resolved imaging and competition assays demonstrated that sticker number governs the kinetics of pyrenoid formation and determines cellular fitness under fluctuating carbon conditions. Our findings establish sticker number as an evolutionary tuning parameter that balances condensate formation, dynamics, and function, providing a quantitative framework for linking the molecular grammar of phase separation to biological fitness.

cell biology↗

bioRxiv2024

SlimVar: rapid in vivo single-molecule tracking of chromatin regulators in plants

Epigenetic regulation occurs over many rounds of cell division in higher organisms. However, visualisation of the regulators in vivo is limited by imaging dynamic molecules deep in tissue. We report a technology--Variable-angle Slimfield microscopy (SlimVar)-- that enables tracking of single fluorescent reporters to 30 {micro}m depth through multiple Arabidopsis thaliana root tip cell layers. SlimVar uses rapid photobleaching to resolve tracked particles to molecular steps in intensity. By modifying widefield microscopy to minimise optical aberrations and robustly post-process few-photon signals, SlimVar mitigates performance losses at depth. We use SlimVar to quantify chromatin-protein assemblies in nuclei, finding that two homologous proteins key to epigenetic switching at FLOWERING LOCUS C (FLC) --cold-induced VERNALISATION INSENSITIVE3 (VIN3) and constitutively expressed VERNALISATION 5 (VRN5)--exhibit dynamic assemblies during FLC silencing. Upon cold exposure, the number of assembly molecules increases up to 100% to a median of [~]20 molecules. Larger VRN5 assemblies preferentially colocalise with an FLC lacO transgenic reporter during prolonged cold and persist after return to warmth. Our findings support a hybrid model of epigenetic memory in which nucleation of histone trimethylation is assisted by dynamic protein assemblies over extended durations. SlimVar offers molecular insights into proteins expressed at physiological levels in tissues.

plant biology↗

bioRxiv2024

Functional specialization of Arabidopsis VEL polymerization domains in the switch to Polycomb silencing

Cold-induced epigenetic silencing of Arabidopsis FLOWERING LOCUS C (FLC) requires the Polycomb Repressive Complex 2 and accessory proteins VIN3 and VRN5. VIN3 and VRN5 interact via head-to-tail VEL polymerization domains, but how these functionally contribute to the switch to an epigenetically silenced state remains poorly understood. Here, we determine that VIN3 VEL polymerization involves higher order nuclear VIN3 assemblies in vivo, promotes strong chromatin association and efficient H3K27me3 nucleation. However, we also show that the polymerization domains of VIN3 and VRN5 are not equivalent: VRN5 VEL domain is not required for silencing despite its role in physically connecting VIN3 with the PRC2 complex and VRN5 VEL is unable to functionally replace VIN3 VEL in vivo. Both VIN3 and VRN5 homologs are present throughout angiosperm species, suggesting a functional requirement for maintaining different polymerization modalities. This work reveals distinct roles for multifunctional polymerization domains of Polycomb accessory proteins underpinning the onset of epigenetic silencing.

plant biology↗