Biology subjects

Collinson, I.

Publications and source records attributed to Collinson, I..

At least 19 recordsLinked to original sources

bioRxiv2025

Intercellular mitochondrial transfer and transmitophagy in response to protein import dysfunction

Mitochondrial protein import is critical for organelle biogenesis and maintenance, essential processes for cellular homeostasis. Import dysfunction compromises cellular energy supplies which is damaging to cells, particularly those with high energetic demands like neurons. Previously, we have shown that import failure is rescued by intercellular mitochondrial transfer via tunnelling nanotubes (TNTs) however, the fate of the transferred mitochondria and the mechanistic basis for rescue was unresolved. Here, we show that bi-directional mitochondrial trafficking between cells harbouring import-defective and import-competent mitochondria has distinct regulation and consequences. Transferred import-defective mitochondria are highly fragmented and destined for canonical lysosomal degradation. In contrast, ROS-producing mitochondria at the periphery of cells with import-competent mitochondria are transferred into neighbouring cells undergoing import failure; these new arrivals then accumulate within previously uncharacterised mitochondrial degradation bodies (MBDs). We speculate that the co-operation of these distinct examples of TNT- mediated conventional and non-canonical transmitophagy instigate mitochondrial regeneration, and thereby rescue.

cell biology↗

bioRxiv2025

Mitochondrial presequences harbor variable strengths to maintain organellar function

Hundreds of mitochondrial-destined proteins rely on N-terminal presequences for organellar targeting and import. While generally described as positively charged amphipathic helices, presequences lack a consensus motif and thus likely promote the import of proteins into mitochondria with variable efficiencies. Indeed, the concept of presequence "strength" critically underlies biological models such as stress sensing, yet a quantitative analysis of what dictates "strong" versus "weak" presequences is lacking. Furthermore, the extent to which presequence strength affects mitochondrial function and cellular fitness remains unclear. Here, we capitalize on the high-throughput and kinetic nature of the MitoLuc mitochondrial protein import assay to quantify multiple aspects of presequence strength. We find that select presequences, including those that regulate the mitochondrial unfolded protein response (UPRmt), are sufficient to impart differential import efficiencies during mitochondrial uncoupling. Surprisingly, we find that presequences beyond those classically associated with stress signaling promote highly variable import efficiency in stressed and basal (i.e., non-stressed) conditions in vitro, suggesting that presequence strength may influence a broader array of processes than currently appreciated. We exploit this variability to demonstrate that only presequences that promote robust import in vitro can fully rescue defects in respiratory growth in Complex IV-deficient yeast, suggesting that presequence strength dictates metabolic potential. Collectively, our findings demonstrate that presequence strength can describe numerous metrics, such as total imported protein, maximal import velocity, or sensitivity to uncoupling, suggesting that the annotation of presequences as "weak" versus "strong" requires more nuanced characterization than is typically performed. Importantly, we find that such variability in presequence strength meaningfully affects cellular fitness in processes beyond stress signaling, suggesting that organisms may broadly exploit presequence strength to fine-tune mitochondrial import and thus organellar homeostasis.

biochemistry↗

bioRxiv2024

Alternative Import-Channels And Destinations Of Mitochondrial PINK1 Controlled By Trans-Membrane-Domain Structural Plasticity

Entry of the PINK1-kinase into the mitochondrial inner-membrane results in cleavage by the rhomboid-protease PARL, followed by retro-translocation back to the outer-membrane and proteasomal-degradation. Failure of this process, in compromised mitochondria, leads to kinase activation at the surface, and ultimately mitophagy. Analysis of PINK1-import within intact cells reveals an alternative pathway into the matrix. Structural modelling predicts that PINK1s trans-membrane-domain (TMD) forms either an -helix or /{beta}-hybrid at the interface between Tim17, of the TIM23-core-complex, and, respectively, either Romo1 or PARL. These mutually exclusive interactions both encapsulate a hydrated protein-channel. The -helical-TMD form adopts a pose suggestive of translocation through the Romo1/Tim17-channel, while the /{beta}-hybrid-TMD is retracted into PARLs active-site for cleavage, presumably after import through the adjacent rhomboid/Tim17-channel. We propose structural plasticity of the PINK1-TMD underlies alternative destinies for full-length matrix-import or cleavage/retro-translocation. The results reveal new insights of PINK1s role in mitochondrial function, quality-control and early-onset Parkinsons disease.

biochemistry↗

bioRxiv2024

A whole cell luminescence-based screen for inhibitors of the bacterial Sec machinery

There is a pressing need for new antibiotics to combat rising resistance to those already in use. The bacterial general secretion (Sec) system has long been considered a good target for novel antimicrobials thanks to its irreplacable role in maintaining cell envelope integrity; yet the lack of a robust, high-throughput method to screen for Sec inhibition has so far hampered efforts to realise this potential. Here, we have adapted our recently-developed in vitro assay for Sec activity - based on the split NanoLuc luciferase - to work at scale and in living cells. A simple counterscreen allows compounds that specifically target Sec to be distinguished from those with other effects on cellular function. As proof of principle we have applied this assay to a library of 5000 compounds and identified a handful of moderately effective in vivo inhibitors of Sec. We therefore anticipate that the methods presented here will be scalable to larger compound libraries, in the ultimate quest for Sec inhibitors with clinically relevant properties.

biochemistry↗

bioRxiv2023

The α-dystroglycan N-terminus is a broad-spectrum antiviral agent against SARS-CoV-2 and enveloped viruses

The COVID-19 pandemic has shown the need to develop effective therapeutics in preparedness for further epidemics of virus infections that pose a significant threat to human health. As a natural compound antiviral candidate, we focused on -dystroglycan, a highly glycosylated basement membrane protein that links the extracellular matrix to the intracellular cytoskeleton. Here we show that the N-terminal fragment of -dystroglycan (-DGN), as produced in E. coli in the absence of post-translational modifications, blocks infection of SARS-CoV-2 in cell culture, human primary gut organoids and the lungs of transgenic mice expressing the human receptor angiotensin I-converting enzyme 2 (hACE2). Prophylactic and therapeutic administration of -DGN reduced SARS-CoV-2 lung titres and protected the mice from respiratory symptoms and death. Recombinant -DGN also blocked infection of a wide range of enveloped viruses including the four Dengue virus serotypes, influenza A virus, respiratory syncytial virus, tick-borne encephalitis virus, but not human adenovirus, a non-enveloped virus in vitro. This study establishes soluble recombinant -DGN as a broad-band, natural compound candidate therapeutic against enveloped viruses.

microbiology↗

bioRxiv2023

Cryo-electron tomography of C. elegans mitochondria reveals how the ATP synthase dimer interface shapes crista membranes

Mitochondrial ATP synthases form rows of dimers, which induce membrane curvature to give cristae their characteristic lamellar or tubular morphology. The angle formed between the central stalks of ATP synthase dimers varies between species. Using cryo-electron tomography and sub-tomogram averaging, we determined the structure of the ATP synthase dimer from the nematode worm C. elegans and show that the dimer angle differs from previously determined structures. The consequences of this species-specific difference at the dimer interface were investigated by comparing C. elegans and S. cerevisiae mitochondrial morphology. We reveal that C. elegans has a larger ATP synthase dimer angle with more lamellar (flatter) cristae when compared to yeast. The underlying cause of this difference was investigated by generating an atomic model of the C. elegans ATP synthase dimer by homology modelling. A comparison of our C. elegans model to an existing S. cerevisiae structure reveals the presence of extensions and rearrangements in C. elegans subunits associated with maintaining the dimer interface. We speculate that increasing dimer angles could provide an advantage for species that inhabit variable-oxygen environments by forming flatter more energetically efficient cristae.

biochemistry↗

bioRxiv2022

Intercellular Mitochondrial Transfer as a Rescue Mechanism in Response to Protein Import Failure

Mitochondria are the powerhouses of eukaryotic cells, composed mostly of nuclear-encoded proteins imported from the cytosol. Thus, problems with the import machinery will disrupt their regenerative capacity and the cells energy (ATP) supplies-particularly troublesome for energy demanding cells like neurons and myocytes. Unsurprisingly then, dysfunctional import is implicated in disease. This study explores the consequences of import failure in mammalian cells; wherein, blocking the import machinery has profound effects on mitochondrial ultra-structure and dynamics, but, surprisingly, does not impact import. The explanation is an astonishing response involving intercellular mitochondrial transfer via tunnelling nanotubes: for the import of healthy mitochondria and jettisoning of those with jammed import sites. These observations support the existence of a widespread mechanism for the rescue of mitochondrial protein import failure. One-Sentence SummaryA mitochondrial import rescue mechanism involving intercellular mitochondrial transport through tunneling nanotubes (TNTs).

cell biology↗

bioRxiv2022

Perturbation of the mitochondrial import machinery by disease prone Tau affects organelle morphology and reduces neuronal complexity

Protein import into mitochondria is an intricate and highly conserved process essential for organellar biogenesis, and maintenance of its structure and function. Defects in the import apparatus impact the assembly of the respiratory chain and ATP synthase complexes required for oxidative phosphorylation, compromising the supply of ATP to the cytosol. The consequences of reduced bioenergetic function are particularly severe for cells with high energetic demands, such as neurons. However, relatively little is known about how defective import contributes to neurodegeneration, or how aggregation prone toxic proteins, characteristic of neurodegenerative disease, impact mitochondrial import efficiency. Here, we used HeLa cells to investigate how expressing Tau, or a disease-causing variant, affects mitochondrial import activity, morphology, and function. We found that a variant associated with frontotemporal dementia (TauP301L), but not the native version, colocalises with mitochondria, associating with TOM40-the protein-channel component of the outer membrane import complex. Interestingly, TauP301L production had no discernible effect on overall mitochondrial import function, despite associating with TOM40 and altering mitochondrial morphology. This raised suspicions of a rescue mechanism manifested by the appearance of microtubule and actin containing tunnelling nanotubes (TNTs), used to recruit healthy mitochondria from neighbouring cells and/ or dispose of mitochondria containing aggregated Tau. Furthermore, in primary neuronal cultures TauP301L induces morphological changes that resemble a neurodegeneration-like phenotype-also mirrored in cells where the import sites are blocked artificially. These results reveal an intriguing link between the production of aggregation prone protein variants, such as TauP301L and others, with the mitochondrial protein import machinery relevant to neurodegenerative disease.

cell biology↗

bioRxiv2022

The NanoLuc Assay System for Accurate Real-Time Monitoring of Mitochondrial Protein Import within Intact Mammalian Cells

Only a few proteins (13 in humans) are encoded by the mammalian mitochondrial genome. Therefore, the other mitochondrial resident proteins (>1000) must be recruited via specialised import pathways. Protein import is critical for mitochondrial biogenesis and bioenergetic function and health; loss of function has been implicated with a wide range of pathologies. Despite this, our understanding of the kinetic and dynamics of import is somewhat limited, particularly within mammalian cells. Here, we report an adaptation of an assay system, established previously to monitor mitochondrial import into isolated yeast mitochondria, to quantitatively monitor mitochondrial import inside mammalian cells. The reporting is based on a split luciferase, whereby the large fragment is segregated in the mitochondrial matrix and the small complementary fragment is fused to the C-terminus of a recombinant precursor protein destined for import. Following import successively through the TOM complex of the outer membrane and the TIM23 complex of the inner membrane, the complementary fragments combine to form an active luciferase. The resultant luminescent signal provides a sensitive, accurate, free of noise and continuous measure of protein import, enabling mathematical model fitting to identify and understand the steps that make up import. This advance allows detailed mechanistic examination of the transport process in live cells. In addition, the assay will enable characterisation of the protein import when the machinery is challenged; for example, in situations associated with disease. Moreover, the assay is compatible with high throughput for large data set collection and kinetic modelling, as well as for drug screening and characterisation. Our set-up also has the potential to be adapted for the analysis of alternative transport systems and different cell types, and even for multicellular model organisms.

biochemistry↗

bioRxiv2022

INTERACTION OF THE PERIPLASMIC CHAPERONE SURA WITH THE INNER MEMBRANE PROTEIN SECRETION (SEC) MACHINERY

Gram-negative bacteria are surrounded by two protein-rich membranes with a peptidoglycan layer sandwiched between them. Together they form the envelope (or cell wall), crucial for energy production, lipid biosynthesis, structural integrity, and for protection against the physical and chemical environmental challenges. To achieve envelope biogenesis, periplasmic and outer-membrane proteins (OMPs) must be transported from the cytosol and through the inner-membrane, via the ubiquitous SecYEG protein-channel. Emergent proteins either fold in the periplasm or cross the peptidoglycan (PG) layer towards the outer-membrane for insertion through the {beta}-barrel assembly machinery (BAM). Trafficking of hydrophobic proteins through the periplasm is particularly treacherous given the high protein density and the absence of energy (ATP or chemiosmotic potential). Numerous molecular chaperones assist in the prevention and recovery from aggregation, and of these SurA is known to interact with BAM, facilitating delivery to the outer-membrane. However, it is unclear how proteins emerging from the Sec-machinery are received and protected from aggregation and proteolysis prior to an interaction with SurA. Through biochemical analysis and electron microscopy we demonstrate the binding capabilities of the unoccupied and substrate-engaged SurA to the inner-membrane translocation machinery complex of SecYEG-SecDF-YidC - aka the holo-translocon (HTL). Supported by AlphaFold predictions, we suggest a role for periplasmic domains of SecDF in chaperone recruitment to the protein translocation exit site in SecYEG. We propose that this immediate interaction with a recruited chaperone helps to prevent aggregation and degradation of nascent envelope proteins, facilitating their safe passage to the periplasm and outer-membrane.

biochemistry↗

bioRxiv2022

A bacterial secretosome for regulated envelope biogenesis and quality control

As the first line of defence against antibiotics, the Gram-negative bacterial envelope and its biogenesis are of considerable interest to the microbiological and biomedical communities. All bacterial proteins are synthesised in the cytosol, so inner- and outer-membrane proteins, and periplasmic residents have to be transported to their final destinations via specialised protein machinery. The Sec translocon, a ubiquitous integral inner-membrane (IM) complex, is key to this process as the major gateway for protein transit from the cytosol to the cell envelope; this can be achieved during their translation, or afterwards. Proteins need to be directed to the inner-membrane (usually co-translational), otherwise SecA utilises ATP and the proton-motive-force (PMF) to drive proteins across the membrane post-translationally. These proteins are then picked up by chaperones for folding in the periplasm or delivered to the {beta}-barrel assembly machinery (BAM) for incorporation into the outer-membrane. The core heterotrimeric SecYEG-complex forms the hub for an extensive network of interactions that regulate protein delivery and quality control. Here, we conduct a biochemical exploration of this secretosome - a very large, versatile and inter-changeable assembly with the Sec-translocon at its core; featuring interactions that facilitate secretion (SecDF), inner- and outer-membrane protein insertion (respectively, YidC and BAM), protein folding and quality control (e.g. PpiD, YfgM and FtsH). We propose the dynamic interplay amongst these and other factors act to ensure efficient whole envelope biogenesis, regulated to accommodate the requirements of cell elongation and division. This organisation would be essential for cell wall biogenesis and remodelling and thus its perturbation would be a good strategy for the development of anti-microbials.

biochemistry↗

bioRxiv2021

Towards a molecular mechanism underlying mitochondrial protein import through the TOM-TIM23 complex

Nearly all mitochondrial proteins need to be targeted for import from the cytosol. For the majority, the first port of call is the translocase of the outer membrane (TOM complex), followed by a procession of alternative molecular machines, conducting transport to their final destination. The pre-sequence translocase of the inner-membrane (TIM23-complex) imports proteins with cleavable pre-sequences, and comes in two flavours: the TIM23SORT complex mediates inner mitochondrial membrane (IMM) protein insertion and the TIM23MOTOR complex delivers proteins to the matrix. Progress in understanding these transport mechanisms has been hampered by the poor sensitivity and time-resolution of import assays. However, with the development of an assay based on split NanoLuc luciferase, we can now explore this process in greater detail. Here, we apply this new methodology to understand how {Delta}{psi} and ATP hydrolysis, the two main driving forces for import through the TIM23MOTOR complex, contribute to the import of pre-sequence-containing precursors (PCPs) with varying properties. Notably, we found that two major rate-limiting steps define PCP import time: passage of PCP across the OMM and initiation of IMM transport by the pre-sequence. The rates of these steps are influenced by PCP properties such as size and net charge, but not total amount of PCP imported - emphasising the importance of collecting rapid kinetic data, achieved here, to elucidate mechanistic detail. The apparent distinction between transport through the two membranes (passage through TOM is substantially complete before PCP-TIM engagement) is in contrast with the current view that import occurs through TOM and TIM in a single continuous step. Our results also indicate that PCPs spend very little time in the TIM23 channel - presumably rapid success or failure of import is critical for maintaining mitochondrial fitness.

biochemistry↗

bioRxiv2021

Maintenance of Complex I and respiratory super-complexes by NDUF-11 is essential for respiratory function, mitochondrial structure and health in C. elegans

Mitochondrial super-complexes form around a conserved core of monomeric complex I and dimeric complex III; wherein subunit NDUFA11, of the former, is conspicuously situated at the interface. We identified B0491.5 (NDUF-11) as the C. elegans homologue, of which animals homozygous for a CRISPR-Cas9 generated knockout allele arrested at the L2 development stage. Reducing expression by RNAi allowed development to the adult stage, enabling characterisation of the consequences: destabilisation of complex I and its super-complexes, and perturbation of respiratory function. The loss of NADH-dehydrogenase activity is compensated by enhanced complex II activity, resulting in excessive detrimental ROS production. Meanwhile, electron cryo-tomography highlight aberrant cristae morphology and widening of the inter-membrane space and cristae junctions. The requirement of NDUF-11 for balanced respiration, mitochondrial morphology and development highlights the importance of complex I/ super-complex maintenance. Their perturbation by this, or other means, is likely to be the cause of metabolic stress and disease.

molecular biology↗

bioRxiv2020

Refined measurement of SecA-driven protein transport reveals indirect coupling to ATP turnover

The universally conserved Sec system is the primary method cells utilise to transport proteins across membranes. Until recently, measuring the activity - a prerequisite for understanding how biological systems works - has been limited to discontinuous protein transport assays with poor time resolution, or used as reporters large, non-natural tags that interfere with the process. The development of an assay based on a split super-bright luciferase (NanoLuc) changed this. Here, we exploit this technology to unpick the steps that constitute post-translational transport in bacteria. Under the conditions deployed, transport of the model pre-protein substrate proSpy occurs at 200 amino acids per minute with the data best fit by a series of large, [~]30 amino acid, steps each coupled to many (100s) ATP hydrolysis events. Prior to that, there is no evidence for a distinct, rate-limiting initiation event. Kinetic modelling suggests that SecA-driven transport activity is facilitated by the substrate (polypeptide) concentration gradient - in keeping with classical membrane transporters. Furthermore, the features we describe are consistent with a non-deterministic motor mechanism, such as a Brownian ratchet.

biochemistry↗

bioRxiv2019

Energy landscape steering in SecYEG mediates dynamic coupling in ATP driven protein translocation

The Sec translocon is a highly conserved membrane complex for transport of polypeptides across, or into, lipid bilayers. In bacteria, the core protein-channel SecYEG resides in the inner-membrane, through which secretion is powered by the cytosolic ATPase SecA. Here, we use single-molecule fluorescence to interrogate the dynamic state of SecYEG throughout the hydrolytic cycle of SecA. We show that the SecYEG channel fluctuates between open and closed states faster ([~]20-fold during transport) than ATP turnover; while the nucleotide status of SecA modulates the rates of opening and closure. Interestingly, a SecY variant (PrlA4), exhibiting faster protein transport, but unaffected ATPase rates, increases the dwell time in the open state, facilitating pre-protein diffusion through the pore; thereby improving the efficiency of translocation. Thus, contrary to prevailing structure-based models, SecYEG plays an integral part in the translocation mechanism through dynamic allosteric coupling in which SecA steers the energy landscape of the protein-channel.

biophysics↗

bioRxiv2019

HDX-MS reveals nucleotide-regulated, anti-correlated opening and closure of SecA and SecY channels of the bacterial translocon

The bacterial Sec translocon is a multi-component protein complex responsible for translocating diverse proteins across the plasma membrane. For post-translational protein translocation, the Sec-channel - SecYEG - associates with the motor protein SecA to mediate the ATP-dependent transport of unfolded pre-proteins across the membrane. Based on the structure of the machinery, combined with ensemble and single molecule analysis, a diffusional based Brownian ratchet mechanism for protein secretion has been proposed [Allen et al. eLife 2016;5:e15598]. However, the conformational dynamics required to facilitate this mechanism have not yet been fully resolved. Here, we employ hydrogen-deuterium exchange mass spectrometry (HDX-MS) to reveal striking nucleotide-dependent conformational changes in the Sec protein-channel. In addition to the ATP-dependent opening of SecY, reported previously, we observe a counteracting, also ATP-dependent, constriction of SecA around the mature regions of the pre-protein. Thus, ATP binding causes SecY to open and SecA to close, while ATP hydrolysis has the opposite effect. This alternating behaviour could help impose the directionality of the Brownian ratchet for protein transport through the Sec machinery, and possibly in translocation systems elsewhere. The results highlight the power of HDX-MS for interrogating the dynamic mechanisms of diverse membrane proteins; including their interactions with small molecules such as nucleotides (ATPases and GTPases) and inhibitors (e.g. antibiotics).

biophysics↗

bioRxiv2019

A ‘proton ratchet’ for coupling the membrane potential to protein transport

Transport of proteins across and into membranes is a fundamental biological process with the vast majority being conducted by the ubiquitous Sec machinery. In bacteria, this is usually achieved when the SecY-complex engages the cytosolic ATPase SecA (secretion) or translating ribosomes (insertion). Great strides have been made towards understanding the mechanism of protein translocation. Yet, important questions remain - notably, the nature of the individual steps that constitute transport, and how the proton-motive force (PMF) across the plasma membrane contributes. Here, we apply a recently developed high-resolution protein transport assay to explore these questions. We find that pre-protein transport is limited primarily by the diffusion of arginine residues across the membrane, particularly in the context of bulky hydrophobic sequences. This specific effect of arginine, caused by its positive charge, is mitigated for lysine which can be deprotonated and transported across the membrane in its neutral form. These observations have interesting implications for the mechanism of protein secretion, suggesting a simple mechanism by which PMF can aid transport, and enabling a proton ratchet, wherein re-protonation of exiting lysine residues prevents channel re-entry, biasing transport in the outward direction.

biochemistry↗

bioRxiv2019

Trans-membrane association of the Sec and BAM complexes for bacterial outer-membrane biogenesis

SUMMARYThe outer-membrane of Gram-negative bacteria is critical for surface adhesion, pathogenicity, antibiotic resistance and survival. The major constituent – hydrophobic β-barrel Outer-Membrane Proteins (OMPs) – are secreted across the inner-membrane through the Sec-translocon for delivery to periplasmic chaperones e.g. SurA, which prevent aggregation. OMPs are then offloaded to the β-Barrel Assembly Machinery (BAM) in the outer-membrane for insertion and folding. We show the Holo-TransLocon (HTL: an assembly of the protein-channel core-complex SecYEG, the ancillary sub-complex SecDF, and the membrane ‘insertase’ YidC) contacts SurA and BAM through periplasmic domains of SecDF and YidC, ensuring efficient OMP maturation. Our results show the trans-membrane proton-motive-force (PMF) acts at distinct stages of protein secretion: for SecA-driven translocation across the inner-membrane through SecYEG; and to communicate conformational changes via SecDF to the BAM machinery. The latter presumably ensures efficient passage of OMPs. These interactions provide insights of inter-membrane organisation, the importance of which is becoming increasingly apparent.Competing Interest StatementThe authors have declared no competing interest.View Full Text

biochemistry↗