Biology subjects

Payne-Dwyer, A. L.

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

6 recordsLinked to original sources

bioRxiv2024

Modular structure of RNA 3' processing condensates involving the Arabidopsis RNA binding protein FCA

Our understanding of the functional requirements underpinning biomolecular condensation in vivo is still relatively poor. The Arabidopsis RNA binding protein FCA is found in liquid-like nuclear condensates that function in transcription termination, promoting proximal polyadenylation at many targets in the Arabidopsis genome. To further understand the properties of these condensates in vivo we used single particle tracking experiments on FCA reporters stably expressed at endogenous levels in plant nuclei. These revealed FCA forms a core oligomer of [~]4 molecules that multimerizes into higher-order particles. This assembles into macromolecular condensates through the function of the coiled-coil protein FLL2, which is genetically required for FCA function. FLL2 predominately co-localizes with FCA in larger sized condensates. A missense mutation in the FCA RRM domain, also genetically required for FCA function, reduced both average FCA particle and condensate size but did not perturb the core oligomer. Our work points to a modular structure for FCA condensates involving multimerization of core oligomers that assemble into functional macromolecular condensates via associated RNA and FLL2 interactions.

molecular biology↗

bioRxiv2023

The role of BST4 in the pyrenoid of Chlamydomonas reinhardtii

In many eukaryotic algae, CO2 fixation by Rubisco is enhanced by a CO2- concentrating mechanism, which utilizes a Rubisco-rich organelle called the pyrenoid. The pyrenoid is traversed by a network of thylakoid-membranes called pyrenoid tubules, proposed to deliver CO2. In the model alga Chlamydomonas reinhardtii (Chlamydomonas), the pyrenoid tubules have been proposed to be tethered to the Rubisco matrix by a bestrophin-like transmembrane protein, BST4. Here, we show that BST4 forms a complex that localizes to the pyrenoid tubules. A Chlamydomonas mutant impaired in the accumulation of BST4 (bst4) formed normal pyrenoid tubules and heterologous expression of BST4 in Arabidopsis thaliana did not lead to the incorporation of thylakoids into a reconstituted Rubisco condensate. Chlamydomonas bst4 mutant did not show impaired growth at air level CO2. By quantifying the non-photochemical quenching (NPQ) of chlorophyll fluorescence, we show that bst4 displays a transiently lower thylakoid lumenal pH during dark to light transition compared to control strains. When acclimated to high light, bst4 had sustained higher NPQ and elevated levels of light-induced H2O2 production. We conclude that BST4 is not a tethering protein, but rather is an ion channel involved in lumenal pH regulation possibly by mediating bicarbonate transport across the pyrenoid tubules. One-sentence summaryIn Chlamydomonas, the pyrenoid-localized bestrophin-like protein BST4 is a putative ion channel involved in pH regulation of the thylakoid lumen, possibly by mediating bicarbonate transport.

plant biology↗

bioRxiv2022

Super-resolved imaging deciphers ligand dependent membrane behaviour of the onco-immunogenic CCR5 receptor.

The ability of tumors to establish a pro-tumorigenic microenvironment is becoming an important point of investigation in the search for new therapeutics. Tumors form microenvironments in part by the education of immune cells attracted via chemotactic axes such as that of CCR5-CCL5. Further, CCR5 upregulation by cancer cells, coupled with its association with pro-tumorigenic features such as drug-resistance and metastasis, has suggested CCR5 as a target for therapeutic inhibition. However, with several conformational "pools" being reported, phenotypic investigations must be capable of unveiling heterogeneity. Addressing this challenge, we performed structured illumination (SIM) and Partially TIRF coupled HILO (PaTCH) microscopy for super-resolution imaging and single-molecule imaging of CCR5 in fixed cells. Determining the positions of super-resolved CCR5 assemblies revealed a non-random spatial orientation. Further, intensity-tracking of assemblies revealed a distribution of molecular stoichiometries indicative of dimeric sub-units independent of CCL5 perturbation. These biophysical methods can provide important insights into the structure and function of onco-immunogenic receptors and a plethora of other biomolecules. HighlightsO_LIWe use SIM and novel PaTCH microscopy for precise bioimaging and single-molecule tracking of receptor protein CCR5 in model cell line C_LIO_LIBy tracking the position of CCR5 assemblies we conclude that they are clustered in the plasma membrane beyond a level expected from a random distribution C_LIO_LIWe use these high-precision data to determine molecular stoichiometries of CCR5 assemblies C_LI

biophysics↗

bioRxiv2021

RecA and RecB: probing complexes of DNA repair proteins with mitomycin C in live Escherichia coli with single-molecule sensitivity

The RecA protein and RecBCD complex are key bacterial components for the maintenance and repair of DNA. RecBCD is a helicase-nuclease that uses homologous recombination to resolve double-stranded DNA breaks. It also facilitates coating of single-stranded DNA with RecA to form RecA filaments, a vital step in the double-stranded break DNA repair pathway. However, questions remain about the mechanistic roles of RecA and RecBCD in live cells. Here, we use millisecond super-resolved fluorescence microscopy to pinpoint the spatial localization of fluorescent reporters of RecA or RecB at physiological levels of expression in individual live Escherichia coli cells. By introducing the DNA crosslinker mitomycin C, we induce DNA damage and quantify the resulting steady state changes in stoichiometry, cellular protein copy number and molecular mobilities of RecA and RecB. We find that both proteins accumulate in molecular hotspots to effect repair, resulting in RecA stoichiometries equivalent to several hundred molecules that assemble largely in dimeric subunits before DNA damage, but form periodic subunits of approximately 3-4 molecules within mature filaments of several thousand molecules. Unexpectedly, we find that the physiologically predominant forms of RecB are not only rapidly diffusing monomers, but slowly diffusing dimers.

biophysics↗

bioRxiv2021

Membraneless organelles formed by liquid-liquid phase separation increase bacterial fitness

Liquid-liquid phase separation is emerging as a crucial phenomenon in several fundamental cell processes. A range of eukaryotic systems exhibit liquid condensates. However, their function in bacteria, which in general lack membrane-bound compartments, remains less clear. Here, we used high-resolution optical microscopy to observe single bacterial aggresomes, nanostructured intracellular assemblies of proteins, to undercover their role in cell stress. We find that proteins inside aggresomes are mobile and undergo dynamic turnover, consistent with a liquid state. Our observations are in quantitative agreement with phase-separated liquid droplet formation driven by interacting proteins under thermal equilibrium that nucleate following diffusive collisions in the cytoplasm. We have discovered aggresomes in multiple species of bacteria, and show that these emergent, metastable liquid-structured protein assemblies increase bacterial fitness by enabling cells to tolerate environmental stresses. One Sentence SummaryBacteria use subcellular proteinaceous liquid droplets to survive stress

biophysics↗

bioRxiv2021

Combining single-molecule super-resolved localization microscopy with fluorescence polarization imaging to study cellular processes

Super-resolution microscopy has catalyzed valuable insights into the sub-cellular, mechanistic details of many different biological processes across a wide range of cell types. Fluorescence polarization spectroscopy tools have also enabled important insights into cellular processes through identifying orientational changes of biological molecules typically at an ensemble level. Here, we combine these two biophysical methodologies in a single home-made instrument to enable the simultaneous detection of orthogonal fluorescence polarization signals from single fluorescent protein molecules used as common reporters on the localization of proteins in cellular processes. These enable measurement of spatial location to a super-resolved precision better than the diffraction-limited optical resolution, as well as estimation of molecular stoichiometry based on the brightness of individual fluorophores. In this innovation we have adapted a millisecond timescale microscope used for single-molecule detection to enable splitting of fluorescence polarization emissions into two separate imaging channels for s- and p-polarization signals, which are imaged onto separate halves of the same high sensitivity back-illuminated CMOS camera detector. We applied this fluorescence polarization super-resolved imaging modality to a range of test fluorescent samples relevant to the study of biological processes, including purified monomeric green fluorescent protein, single combed DNA molecules, and protein assemblies and complexes from live Escherichia coli and Saccharomyces cerevisiae cells. Our findings are qualitative but demonstrate promise in showing how fluorescence polarization and super-resolved localization microscopy can be combined on the same sample to enable simultaneous measurements of polarization and stoichiometry of tracked molecular complexes, as well as the translational diffusion coefficient.

biophysics↗