Biology subjects

Wilkinson, K.

Publications and source records attributed to Wilkinson, K..

12 recordsLinked to original sources

bioRxiv2026

MFF SUMOylation regulates neuronal mitochondrial morphology and facilitates AMPK-induced mitochondrial fragmentation

Mitochondrial dysfunction and fragmentation in neurons are characteristics of many neurodegenerative conditions. AMP-activated protein kinase (AMPK) is a cellular energy sensor which is hyperactivated in Alzheimers disease. Under stress conditions, AMPK-mediates phosphorylation of the outer mitochondrial membrane protein mitochondrial fission factor (MFF), leading to SUMOylation at Lys151. MFF SUMOylation promotes fission and subsequent mitochondrial fragmentation. Here, using primary neuronal culture, we investigate the role of MFF SUMOylation in mitochondrial morphology and function. We show that MFF SUMOylation is required to maintain neuronal mitochondrial length under basal conditions in axons and dendrites. Using the AMPK activator (AICAR) to induce mitochondrial fission, show that preventing MFF SUMOylation reduces AICAR-induced fragmentation of dendritic mitochondria. These findings reconcile observations of AMPK hyperactivation and mitochondrial fragmentation by revealing the AMPK-MFF-SUMO pathway to be crucial in neuronal mitochondrial fission.

neuroscience↗

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

PINK1 SUMOylation regulates basal mitophagy

The mitochondrial kinase PTEN-induced kinase 1 (PINK1) initiates mitophagy in response to mitochondrial damage, yet mechanisms that constrain its activation under basal conditions remain incompletely defined. Here, we identify a non-canonical SUMO-2/3 modification of PINK1 that suppresses basal mitophagy. We show that the mitochondrial E3 ligase mitochondrial-anchored protein ligase (MAPL/MUL1) promotes PINK1 SUMO-2/3 modification, while MAPL depletion reduces SUMOylation and enhances basal mitophagy. Targeted deSUMOylation increases PINK1 stabilisation and augments mitophagy. These findings reveal a novel SUMO-based checkpoint for basal PINK1 activation and mitochondrial quality control.

cell biology↗

bioRxiv2024

Mapping the endosomal proximity proteome reveals Retromer as a hub for RAB GTPase regulation.

Endosomal retrieval and recycling of integral cargo proteins is essential for cell, tissue and organism-level development and homeostasis and is orchestrated through a specialised retrieval sub-domain on the endosomal vacuole. However, although sub-domain dysfunction is associated with human disease our appreciation of the molecular details and functional components of the retrieval sub-domain(s) remains poorly described. Here, using comparative proximity proteomics of critical retrieval sub-domain components Retromer and Retriever, their cargo adaptors, and a component of the opposing ESCRT-degradative sub-domain, we provide a data-rich resource that identifies new molecular details of retrieval sub-domain composition and organization, including an unrecognised complexity in the interface of Retromer with RAB GTPases. Combining X-ray crystallography and in silico predictions with extensive biochemical and cellular analysis, we dissect the direct association of Retromer with RAB10 regulators DENND4A, DENND4C, TBC1D1, and TBC1D4, and the RAB35 regulator TBC1D13. Overall, we conclude that the Retromer retrieval sub-domain constitutes a major hub for the regulated switching of selected RAB GTPases and propose that this constitutes a major component of the role of Retromer in neuroprotection.

cell biology↗

bioRxiv2023

Coordinated interplay between palmitoylation, phosphorylation and SUMOylation regulates kainate receptor surface expression.

Kainate receptors (KARs) are key regulators of neuronal excitability and synaptic transmission. KAR surface expression is tightly controlled in part by post-translational modifications (PTMs) of the GluK2 subunit. We have shown previously that agonist activation of GluK2-containing KARs leads to phosphorylation of GluK2 at S868, which promotes subsequent SUMOylation at K886 and receptor endocytosis. Furthermore, GluK2 has been shown to be palmitoylated. However, how the interplay between palmitoylation, phosphorylation and SUMOylation orchestrate KAR trafficking remains unclear. Here, we used a library of site-specific GluK2 mutants to investigate the interrelationship between GluK2 PTMs, and their impact on KAR surface expression. We show that GluK2 is basally palmitoylated and that this is decreased by kainate stimulation. Moreover, a non-palmitoylatable GluK2 mutant (C858/C871A) shows enhanced S868 phosphorylation and K886 SUMOylation under basal conditions and is insensitive to KA-induced internalisation. These results indicate that GluK2 palmitoylation contributes to stabilising KAR surface expression and that dynamic depalmitoylation promotes downstream phosphorylation and SUMOylation to mediate activity-dependent KAR endocytosis. Significance StatementPost-translational modifications (PTMs) are biochemical switches that control substrate protein properties and interactions. In consequence, PTMs are critical regulators of essentially all cellular pathways and are vital for eukaryotic cell survival. In the brain, among other roles, PTMs influence neuronal growth, differentiation, synaptic activity and plasticity. Kainate receptors (KARs) play core roles in all these processes and previous work has shown that the GluK2 subunit of KARs is subject to multiple PTMs. Here, using GluK2 as an exemplar protein, we delineate the sequence, coordination, and consequences of the PTMs palmitoylation, phosphorylation and SUMOylation on KAR surface expression. Our data show how the complex interplay between PTMs dynamically regulates synaptic proteins and neuronal function.

neuroscience↗

bioRxiv2023

SUMOylation of MFF is required for stress-induced mitochondrial fission

Mitochondrial fission regulates mitochondrial morphology, function, mitophagy and apoptosis. Fission is mediated by the GTPase dynamin related protein-1 (DRP1) and its recruitment to the outer mitochondrial membrane by DRP1 receptors. Mitochondrial fission factor (MFF) is considered the major pro-fission receptor, whereas the mitochondrial dynamics proteins (MiD49/51) sequester inactive DRP1 and facilitate the MFF-DRP1 interaction by forming a trimeric DRP1-MiD-MFF complex. Here, we identify MFF as a target of poly-SUMOylation at a single residue (Lys151). Following bioenergetic stress, AMPK phosphorylates MFF to promote its SUMOylation, a critical step in stress-induced fragmentation. MFF SUMOylation is not required for DRP1 recruitment from the cytosol but causes a rearrangement of the trimeric fission complex to displace MiD proteins. This alleviates MiD inhibition of DRP1 to facilitate formation of a fission-competent complex. Thus, our data demonstrate that MFF SUMOylation fine-tunes the ratio of MiD to DRP1 for the dynamic control of stress-induced mitochondrial fragmentation.

cell biology↗

bioRxiv2022

GluK2 Q/R editing regulates kainate receptor signalling and synaptic AMPA receptor expression and function.

Q/R editing of the kainate receptor (KAR) subunit GluK2 radically alters properties of recombinant KARs, but the effects in vivo remain largely unexplored. We compared GluK2 editing-deficient mice that express [~]95% unedited GluK2(Q) to wild-type counterparts that express [~]85% edited GluK2(R). At mossy fibre-CA3 (MF-CA3) synapses GluK2(Q) mice displayed enhanced postsynaptic KAR function and increased KAR-mediated presynaptic facilitation, demonstrating heightened ionotropic function. Conversely, there was reduced metabotropic KAR function, assessed by KAR-mediated afterhyperpolarization currents, in GluK2(Q) mice. GluK2(Q) mice had fewer GluA1- and GluA3-containing AMPA receptors (AMPARs) and reduced postsynaptic AMPAR currents at both MF-CA3 and CA1-Schaffer collateral synapses. Moreover, long-term potentiation of AMPAR-mediated transmission at CA1-Schaffer collateral synapses was reduced in GluK2(Q) mice. These findings suggest that GluK2 Q/R editing influences ionotropic/metabotropic balance of KAR signalling to regulate synaptic expression of AMPARs and plasticity.

neuroscience↗

bioRxiv2020

Sustained postsynaptic kainate receptor activation downregulates AMPA receptor surface expression and induces hippocampal LTD

Here we report that sustained activation of GluK2 subunit-containing kainate receptors leads to AMPA receptor endocytosis and a novel form of long-term depression (KAR-LTDAMPAR) in hippocampal neurons. The KAR-evoked loss of surface AMPA receptors requires KAR channel activity and is occluded by the blockade of PKC or PKA. Moreover, in acute hippocampal slices, kainate invoked LTD of AMPA EPSCs. These data, together with our previously reported KAR-LTPAMPAR, demonstrate that KARs bidirectionally regulate synaptic AMPARs and synaptic plasticity.

neuroscience↗

bioRxiv2019

Protective role of the deSUMOylating enzyme SENP3 in myocardial ischaemia-reperfusion injury

Interruption of blood supply to the heart is a leading cause of death and disability. However, the molecular events that occur during heart ischaemia, and how these changes prime consequent cell death upon reperfusion, are poorly understood. Protein SUMOylation is a post-translational modification that has been strongly implicated in the protection of cells against a variety of stressors, including ischaemia-reperfusion. In particular, the SUMO2/3-specific protease SENP3 has emerged as an important determinant of cell survival after ischaemic infarct. Here, we used the Langendorff perfusion model to examine changes in the levels and localisation of SUMOylated target proteins and SENP3 in whole heart. We observed a 50% loss of SENP3 from the cytosolic fraction of hearts after preconditioning, a 90% loss after ischaemia and an 80% loss after ischaemia-reperfusion. To examine these effects further, we performed ischaemia and ischaemia-reperfusion experiments in the cardiomyocyte H9C2 cell line. Similar to whole hearts, ischaemia induced a decrease in cytosolic SENP3. Furthermore, shRNA-mediated knockdown of SENP3 led to an increase in the rate of cell death upon reperfusion. Together, our results indicate that cardiac ischaemia dramatically alter levels of SENP3 and suggest that this may a mechanism to promote cell survival after ischaemia-reperfusion in heart.

cell biology↗

bioRxiv2019

Parkin-mediated ubiquitination contributes to the constitutive turnover of mitochondrial fission factor (Mff)

The mitochondrial outer membrane protein Mitochondrial Fission Factor (Mff) plays a key role in both physiological and pathological fission. It is well established that in stressed or functionally impaired mitochondria the PINK1 recruits the ubiquitin ligase Parkin which ubiquitinates Mff to facilitate the removal of defective mitochondria and maintain the integrity mitochondrial network. Here we show that, in addition to this clearance pathway, Parkin also ubiquitinates Mff in a PINK1-dependent manner under basal, non-stressed conditions to regulate constitutive Mff turnover. We further show that removing Parkin with shRNA knockdown does not completely prevent Mff ubiquitination under these conditions indicating that at least one other ubiquitin ligase contributes to Mff proteostasis. These data demonstrate that Parkin plays a role in physiological maintenance of mitochondrial membrane protein composition in healthy mitochondria through constitutive low-level activation.

cell biology↗

bioRxiv2018

The C-terminal Helix 9 motif regulates cannabinoid receptor type 1 trafficking and surface expression

Cannabinoid type 1 receptor (CB1R) is only stably surface expressed in axons, where it downregulates neurotransmitter release. How this tightly regulated axonal surface polarity is established and maintained is unclear. To address this question, we used time-resolved imaging to determine the trafficking of CB1R from biosynthesis to mature polarised localisation. We show that the secretory pathway delivery of CB1R is axonally biased and that surface expressed CB1R is more stable in axons than in dendrites. This dual mechanism is mediated by the CB1R C-terminal and involves the Helix 9 (H9) domain. Removal of the H9 domain increases dendrite secretory pathway delivery and decreases in surface stability. Furthermore, CB1R{Delta}H9 is more sensitive to agonist-induced internalisation and less efficient at downstream signalling than CB1RWT. Together, these results shed new light on how polarity of CB1R is mediated and indicate that the C-terminal H9 domain plays key roles in this process.

neuroscience↗

bioRxiv2018

ADAR2-mediated Q/R editing of GluK2 regulates kainate receptor upscaling in response to suppression of synaptic activity

Kainate receptors (KARs) regulate neuronal excitability and network function. Most KARs contain the subunit GluK2 and the properties of these receptors are determined in part by ADAR2-mediated mRNA editing of GluK2 that changes a genomically encoded glutamine (Q) to arginine (R). Suppression of synaptic activity reduces ADAR2-dependent Q/R editing of GluK2 with a consequential increase in GluK2-containing KAR surface expression. However, the mechanism underlying this reduction in GluK2 editing has not been addressed. Here we show that induction of KAR upscaling results in proteasomal degradation of ADAR2, which reduces GluK2 Q/R editing. Because KARs incorporating unedited GluK2(Q) assemble and exit the ER more efficiently this leads to an upscaling of KAR surface expression. Consistent with this, we demonstrate that partial ADAR2 knockdown phenocopies and occludes KAR upscaling. Moreover, we show that although the AMPAR subunit GluA2 also undergoes ADAR2-dependent Q/R editing, this process does not mediate AMPAR upscaling. These data demonstrate that activity-dependent regulation of ADAR2 proteostasis and GluK2 Q/R editing are key determinants of KAR, but not AMPAR, trafficking and upscaling.\n\nSummary statementSynaptic suppression promotes proteasomal degradation of the mRNA-editing enzyme ADAR2. Decreased ADAR2 levels reduce Q/R editing of the kainate receptor subunit GluK2 leading to enhanced surface expression and homeostatic upscaling.

neuroscience↗