Biology subjects

Stevens, T. J.

Publications and source records attributed to Stevens, T. J..

12 recordsLinked to original sources

bioRxiv2026

Integration of SYT1 Interactomics and Dual-Localization Proteomics Links ER-PM Contacts to Lignin Deposition

Membrane contact sites (MCSs) are evolutionarily conserved intracellular nanodomains that physically bridge opposing lipid bilayers to facilitate non-vesicular communication and maintain cellular homeostasis. In plants, endoplasmic reticulum-plasma membrane (ER-PM) contact sites play fundamental roles in environmental adaptation, and are populated by specialized proteins which act as tethers such as Synaptotagmin 1 (SYT1). However, a comprehensive view of the molecular machinery governing processes at these junctions is still needed. In this work, we integrate affinity purification mass spectrometry, TurboID proximity labeling, and a dual-localization reanalysis of HyperLOPIT spatial proteomics to functionally map the protein interaction landscape of the ER-PM contact sites protein SYT1. Beyond recovering established ER-PM MCS functions, our analysis identified uncharacterized proteins as bona fide resident components of these junctions, and revealed that these nanodomains act as docking platforms that anchor the monolignol biosynthetic complex. By spatially organizing Membrane Steroid Binding Proteins and cytochrome P450 enzymes, our findings support a model where SYT1-mediated anchoring of this metabolon to ER-PM contact sites optimizes monolignol export required for stress-induced lignification. Ultimately, this proteomic framework expands the functional repertoire of ER-PM contact sites, opening new avenues to uncover hidden roles of MCSs across diverse eukaryotic systems.

molecular biology↗

bioRxiv2024

Identification of Cellular Signatures Associated with Chinese Hamster Ovary (CHO) Cell Adaptation for Secretion of Antibodies

The secretory capacity of Chinese hamster ovary (CHO) cells remains a fundamental bottleneck in the manufacturing of protein-based therapeutics. Unconventional biological drugs with complex structures and processing requirements are particularly problematic. Although engineered vector DNA elements can achieve rapid and high-level therapeutic protein production, a high metabolic and protein folding burden is imposed on the host cell. Cellular adaptations to these conditions include differential gene expression profiles that can in turn influence the productivity and quality control of recombinant proteins. In this study, we used quantitative transcriptomics and proteomics analyses to investigate how biological pathways change with antibody titre. Gene and protein expression profiles of CHO pools and clones producing a panel of different monoclonal and bispecific antibodies were analysed during fed-batch production. Antibody-expressing CHO pools were heterogeneous, resulting in few discernible genetic signatures. Clonal lines derived from these pools, selected for high and low production, yielded a small number of differentially expressed proteins that correlated with productivity and were shared across biotherapeutics. However, the dominant feature associated with higher protein production was transgene copy number and resulting mRNA expression level. Moreover, variability between clones suggested that the process of cellular adaptation is variable with diverse cellular changes associated with individual adaptation events.

bioengineering↗

bioRxiv2023

Cargo selective vesicle tethering: the structural basis for binding of specific cargo proteins by the Golgi tether component TBC1D23

For accurate membrane traffic it is essential that vesicles and other carriers tether and fuse to only the correct compartment. The TGN-localised golgins golgin-97 and golgin-245 capture transport vesicles arriving from endosomes via the protein TBC1D23 that forms a bridge between the golgins and endosome-derived vesicles. The C-terminal domain of TBC1D23 is responsible for vesicle capture, but how it recognises a specific type of vesicle was unclear. A search for binding partners of the C-terminal domain surprisingly revealed direct binding to carboxypeptidase D (CPD) and syntaxin-16, both known cargo proteins of the captured vesicles. Binding is via a TLY-containing sequence present in both proteins. A crystal structure reveals how this "acidic TLY motif" binds to the C-terminal domain of TBC1D23. An acidic TLY motif is also present in the tails of other endosome-to-Golgi cargo, and these also bind TBC1D23. Structure-guided mutations in the C-terminal domain that disrupt motif binding in vitro also block vesicle capture in vivo. Thus, TBC1D23 attached to golgin-97 and golgin-245 captures vesicles by a previously undescribed mechanism: the recognition of a motif shared by cargo proteins carried by the vesicle. One sentence summaryA class of transport vesicle destined for the Golgi is recognized by a tether binding directly to the cargo it is carrying.

cell biology↗

bioRxiv2023

Enhancer-promoter interactions are reconfigured through the formation of long-range multiway chromatin hubs as mouse ES cells exit pluripotency

Enhancers are genomic DNA sequences that bind transcription factors, chromatin regulators and non-coding transcripts to modulate the expression of target genes. They have been found to act from different locations relative to a gene and to modulate the activity of promoters up to [~]1 Mb away. Here we report the first 3D genome structures of single mouse ES cells as they are induced to exit pluripotency, transition through a formative stage and undergo neuroectodermal differentiation. In order to directly study how interactions between enhancers and promoters are reconfigured genome wide we have determined 3D structures of haploid cells using single cell Hi-C, where we can unambiguously map the contacts to particular chromosomes. We find that there is a remarkable reorganisation of 3D genome structure where inter-chromosomal intermingling increases dramatically in the formative state. This intermingling is associated with the formation of a large number of multiway hubs that bring together enhancers and promoters with similar chromatin states from typically 5-8 distant chromosomal sites that are often separated by many Mb from each other. Genes important for pluripotency exit establish contacts with emerging enhancers within multiway chromatin hubs as cells enter the formative state, consistent with these structural changes playing an important role in establishing new cell identities. Furthermore, we find that different multiway chromatin hubs, and thus different enhancer-promoter interactions, are formed in different individual cells. Our results suggest that studying genome structure in single cells will be important to identify key changes in enhancer-promoter interactions that occur as cells undergo developmental transitions.

developmental biology↗

bioRxiv2022

De novo design of modular peptide binding proteins by superhelical matching

General approaches for designing sequence-specific peptide binding proteins would have wide utility in proteomics and synthetic biology. Although considerable progress has been made in designing proteins which bind to other proteins, the general peptide binding problem is more challenging as most peptides do not have defined structures in isolation, and to offset the loss in solvation upon binding the protein binding interface has to provide specific hydrogen bonds that complement the majority of the buried peptides backbone polar groups (1-3). Inspired by natural repeat protein-peptide complexes, and engineering efforts to alter their specificity (4-11), we describe a general approach for de novo design of proteins made out of repeating units that bind peptides with repeating sequences such that there is a one to one correspondence between repeat units on the protein and peptide. We develop a rapid docking plus geometric hashing method to identify protein backbones and protein-peptide rigid body arrangements that are compatible with bidentate hydrogen bonds between side chains on the protein and the backbone of the peptide (12); the remainder of the protein sequence is then designed using Rosetta to incorporate additional interactions with the peptide and drive folding to the desired structure. We use this approach to design, from scratch, alpha helical repeat proteins that bind six different tripeptide repeat sequences--PLP, LRP, PEW, IYP, PRM and PKW--in near polyproline 2 helical conformations. The proteins are expressed at high levels in E. coli, are hyperstable, and bind peptides with 4-6 copies of the target tripeptide sequences with nanomolar to picomolar affinities both in vitro and in living cells. Crystal structures reveal repeating interactions between protein and peptide interactions as designed, including a ladder of protein sidechain to peptide backbone hydrogen bonds. By redesigning the binding interfaces of individual repeat units, specificity can be achieved for non-repeating sequences, and for naturally occuring proteins containing disordered regions. Our approach provides a general route to designing specific binding proteins for a broad range of repeating and non-repetitive peptide sequences.

biochemistry↗

bioRxiv2022

Zasp52 strengthens whole embryo tissue integrity through supracellular actomyosin networks

During morphogenesis, large scale changes of tissue primordia occur that are all coordinated across an embryo. In Drosophila, several tissue primordia and embryonic regions are bordered or encircled by supracellular actomyosin cables, junctional actomyosin enrichments networked between many neighbouring cells. We show that the single Drosophila Alp/Enigma family protein Zasp52, which is most prominently found in Z-discs of muscles, is a component of many supracellular actomyosin structures during embryogenesis, including the ventral midline and the boundary of the salivary gland placode. Zasp52, we uncover, contains within its central coiled-coil region a type of actin-binding motif usually found in CapZbeta proteins, and this domain displays actin binding activity. Using endogenously-tagged lines we identify that Zasp52 interacts with a subset of junctional components, including APC2, Polychaetoid/ZO-1, and Sidekick, as well as actomyosin regulators. Analysis of zasp52 mutant embryos revealed that the severity of the embryonic defects observed scales inversely with the amount of functional protein left. Large tissue deformations occur at sites where actomyosin cables are found during embryogenesis, suggesting a model whereby supracellular cables containing Zasp52 aid to insulate morphogenetic changes from one another. This is further supported by the analysis of in vivo processes as well as by an in silico vertex model of morphogenetic domains separated by a stiffness boundary.

developmental biology↗

bioRxiv2022

Functional unknomics: closing the knowledge gap to accelerate biomedical research

The human genome encodes [~]20,000 proteins, many still uncharacterised. Scientific and social factors have resulted in a focus on well-studied proteins, leading to a concern that poorly understood genes are unjustifiably neglected. To address this, we have developed an "Unknome database" that ranks proteins based on how little is known about them. We applied RNAi in Drosophila to 260 unknown genes that are conserved between flies and humans. About a quarter are required for viability, and functional screening of the rest revealed hits for fertility, development, locomotion, protein quality control and resilience to stress. CRISPR/Cas9 gene disruption validated a component of Notch signalling and two genes contributing to male fertility. Our work demonstrates the importance of poorly understood genes, provides a resource for future research acceleration, and highlights a need for our awareness of ignorance to be protected from erosion by automated database annotation.

molecular biology↗

bioRxiv2021

GOLPH3 and GOLPH3L are broad-spectrum COPI adaptors for sorting into intra-Golgi transport vesicles

Glycosylation is a diverse and abundant modification of proteins, lipids and RNA. The fidelity of glycosylation is, in part, assured by the correct compartmentalisation of Golgi-resident glycosylation enzymes within the Golgi stack. The COPI adaptor GOLPH3 has been shown to interact with the cytoplasmic tails of a subset of Golgi enzymes and direct their retention in the Golgi. However, other mechanisms of retention, and other roles for GOLPH3, have been proposed, and a comprehensive characterisation of the clientele of GOLPH3 and its paralogue GOLPH3L has been lacking. The role of GOLPH3 is of particular interest as it is frequently amplified in several solid tumour types. Here, we combine two orthogonal proteomic analyses to identify a diverse range of GOLPH3+3L clients and find that they act in a wide spectrum of glycosylation pathways, or have other roles in the Golgi. Using binding studies, bioinformatics and an in vivo Golgi retention assay, we show that GOLPH3+3L interact with the cytoplasmic tails of their clients through membrane-proximal positively-charged residues. Furthermore, deletion of GOLPH3+3L causes diverse defects in glycosylation. Thus, GOLPH3+3L are major COPI adaptors that impinge on most, if not all, of the glycosylation pathways of the Golgi.

cell biology↗

bioRxiv2020

A subcellular atlas of Toxoplasma reveals the functional context of the proteome

Apicomplexan parasites cause major human disease and food insecurity. They owe their considerable success to novel, highly specialized cell compartments and structures. These adaptations drive their recognition and non-destructive penetration of hosts cells and the elaborate reengineering of these cells to promote growth, dissemination, and the countering of host defenses. The evolution of unique apicomplexan cellular compartments is concomitant with vast proteomic novelty that defines these new cell organizations and their functions. Consequently, half of apicomplexan proteins are unique and uncharacterized, and these cells are, therefore, very poorly understood. Here, we determine the steady-state subcellular location of thousands of proteins simultaneously within the globally prevalent apicomplexan parasite Toxoplasma gondii. This provides unprecedented comprehensive molecular definition to these cells and their novel compartments, and these data reveal the spatial organizations of protein expression and function, adaptation to hosts, and the underlying evolutionary trajectories of these pathogens.

cell biology↗

bioRxiv2020

Live-cell 3D single-molecule tracking reveals how NuRD modulates enhancer dynamics

Enhancer-promoter dynamics are crucial for the spatiotemporal control of gene expression, but it remains unclear whether these dynamics are controlled by chromatin regulators, such as the nucleosome remodelling and deacetylase (NuRD) complex. The NuRD complex binds to all active enhancers to modulate transcription and here we use Hi-C experiments to show that it blurs TAD boundaries and increases the proximity of intermediate-range (~1 Mb) genomic sequences and enhancer-promoter interactions. To understand whether NuRD alters the dynamics of 3D genome structure, we developed an approach to segment and extract key biophysical parameters from 3D single-molecule trajectories of the NuRD complex determined using live-cell imaging. Unexpectedly, this revealed that the intact NuRD complex decompacts chromatin structure and makes NuRD-bound enhancers move faster, increasing the overall volume of the nucleus that these key regulatory regions explore. Interestingly, we also uncovered a rare fast-diffusing state of NuRD bound enhancers that exhibits directed motion. The NuRD complex reduces the amount of time that enhancers remain in this fast-diffusing state, which we propose might otherwise re-organise enhancer-promoter proximity. Thus, we uncover an intimate connection between a chromatin remodeller and the spatial dynamics of the local regions of the genome to which it binds.

cell biology↗

bioRxiv2019

Rho-kinase planar polarisation at tissue boundaries depends on phospho-regulation of membrane residence time

Rho-kinase (Rok) is a major myosin II activator during morphogenesis. In the Drosophila embryonic salivary gland placode Rok is planar polarised at the tissue boundary, through a negative regulation by the apical polarity protein Crumbs that is anisotropically localised at the boundary. However, in inner cells of the placode both Crumbs and Rok are isotropically enriched at junctions. We propose a model that reconciles both behaviours through modulation of Rok membrane residence time by Crumbs and downstream effectors. Using FRAP in embryos expressing endogenously-tagged Rok combined with in silico simulations, we find that the lower membrane dissociation rate (koff) of Rok at the tissue boundary, where Crumbs membrane levels are lower, explains this boundary-specific effect. The S/T-kinase Pak1 negatively affects Rok membrane association in vivo within the epidermis, and in vitro can phosphorylate Rok near the PH domain that mediates membrane association. Pak1 is recruited to the membrane by Cdc42 which, like its binding partner Crumbs, shows anisotropic localisation at the boundary. These data reveal an important mechanism of modulation of Rok membrane residence time via affecting the koff that may be widely employed during tissue morphogenesis.

developmental biology↗

bioRxiv2018

Triparental inheritance in Dictyostelium

Sex promotes the recombination and reassortment of genetic material and is prevalent across eukaryotes. In social amoebae sex involves a promiscuous mixing of cytoplasm before zygotes consume the majority of cells. We report here the first genomewide characterisation of meiotic progeny in Dictyostelium discoideum. We find that recombination occurs at high frequency in pairwise crosses between all three mating types, despite the absence of the SPO11 enzyme that is normally required to initiate crossover formation. In crosses involving three strains, transient fusions involving more than two gametes frequently lead to triparental inheritance, with recombined nuclear haplotypes inherited from two parents and the mitochondrial genome from a third. Cells that do not contribute genetically to the Dictyostelium zygote nucleus thereby have a stake in the next haploid generation. We suggest that this lateral transfer helps to enforce cooperation in this confictual system.

genetics↗