Biology subjects

Heesom, K.

Publications and source records attributed to Heesom, K..

16 recordsLinked to original sources

bioRxiv2026

Motile ciliophagy promotes ciliary recycling under stress

Motile cilia on eukaryotic cell surfaces are exposed to a wide array of external stresses. Ciliary remodeling in response to stress remains understudied despite being pivotal to cell homeostasis. Here, we apply a commonly used calcium perturbation to mimic hyper-osmotic cellular stress in the model ciliate Tetrahymena thermophila. We find that partially deciliated Tetrahymena, upon calcium shock, internalise entire ciliary axonemes into ring-like configurations we refer to as c-rings. Partially deciliated cells recover and regenerate new cilia over time. We propose that the recycling of c-rings releases axonemal building blocks to allow ciliary regeneration. We use time-course ultrastructure expansion microscopy (U-ExM) to show that the ciliary tubulin code undergoes a dynamic pattern of erasure during the bulk disassembly of doublet microtubules within the c-rings. Using whole cell quantitative proteomics, we find that partial deciliation induces the autophagy of c-rings and activates de novo dynein synthesis by upregulating several axonemal dynein assembly factors (DNAAFs). Transmission electron microscopy (TEM) and confocal imaging confirms that intact ciliary axonemes are encapsulated within VPS13A +ve autophagic vacuoles. We propose that internalised motile cilia undergo a regulated process of bulk degradation we term "motile ciliophagy" for the recycling of axonemal components for cilia regeneration. Our work establishes a link between ciliary turnover via macroautophagy and cellular homeostasis which may be a conserved response to stress.

cell biology↗

bioRxiv2026

RUNX1 aberrations in blast-phase CML induce the RBP SPATS2L which promotes growth, survival and stress granule assembly

The RUNX1 transcription factor is a critical regulator of hematopoiesis and frequently mutated in myeloid malignancies. In the myeloproliferative neoplasm, chronic myeloid leukemia (CML), secondary somatic RUNX1 mutations and RUNX1::MECOM/EVI1, are associated with tyrosine kinase inhibitor (TKI) resistance and progression to the blast-phase (BP-CML). Research has predominantly focussed on transcriptional dysregulation mediated by RUNX1 mutations in myeloid malignancies, whilst post-transcriptional dysregulation remains comparatively unexplored. To address this, we used orthogonal organic phase separation (OOPS), to characterise the RNA-binding proteome of RUNX1 deficient BP-CML cells. RUNX1 depleted BP-CML cells exhibited significant alterations to RBP abundance involved in stress response pathways and translation/ribosome-biogenesis (RiBi). Furthermore, RUNX1 depletion or expression of RUNX1::EVI1 in BP-CML cells induced expression and RNA binding activity of SPATS2L, a component of stress granules (SG); membraneless cytoplasmic condensates protecting mRNAs from degradation, promoting survival under stress. Whilst RUNX1 depletion increased SG-assembly, SPATS2L depletion reduced SG-assembly in BP-CML cells and inhibited the growth and survival of multiple BP-CML cell lines. The translation inhibitor homoharringtonine (HHT), used historically in TKI-resistant CML, ablated SG-assembly in BP-CML cells with RUNX1 depletion, and, primary BP-CML cells with LOF/hypomorphic RUNX1 mutations (characterised by defective DNA-binding/CBF{beta}-interaction) were preferentially sensitised to HHT. Finally, suppressing SPATS2L expression induced by RUNX1 depletion, increased the HHT-sensitivity of RUNX1 depleted BP-CML cells, suggesting SPATS2L contributes to therapeutic resistance in CML with RUNX1 mutations. This study suggests that SPATS2L and SG induction could be critical to RUNX1-mutant leukemias, and, provides preliminary evidence for a mutationally-targeted approach in CML with RUNX1 aberrations.

cancer biology↗

bioRxiv2026

FDXR Upregulation by p63/p73 is a Prognostic and Therapeutic Marker of HTLV-1-Associated Adult T Cell Leukemia/Lymphoma

Infection with human T-lymphotropic virus 1 (HTLV-1) can lead to severe diseases, including adult T-cell leukemia/lymphoma (ATL), for which current therapies are inadequate. HTLV-1 persists chronically through integration of its genome into host DNA and expansion of infected clones via cellular proliferation. Selective elimination of infected cells is the ideal treatment goal but requires identifying cellular markers dysregulated by HTLV-1 that can be exploited therapeutically. Combining metabolomics and proteomics analyses, we identified increased expression of the mitochondrial enzyme ferredoxin reductase (FDXR) as a hallmark of HTLV-1-infected cells. In patient cells, FDXR upregulation mirrored disease severity, was enriched in proliferating tumour cells and predicted worse ATL prognosis. Mechanistically, FDXR upregulation was directly driven by the transactivating isoforms of two p53 family members, TAp63 and TAp73. In silico drug susceptibility predictions, followed by targeted cytotoxicity, proliferation and Drug-Seq assays demonstrated that FDXR upregulation is associated with increased sensitivity of ATL cells to repurposed chemotherapeutic agents, particularly fluorouracil. Overall, our findings establish FDXR as a novel biomarker of HTLV-1-associated disease progression and ATL prognosis, elucidate its upstream molecular regulation, and highlight its predictive potential for chemotherapeutic sensitivity of ATL cells.

microbiology↗

bioRxiv2025

TOE1 influences canonical Wnt signalling in myeloid leukaemia cells through LEF-1 modulation and regulates the proliferation of haematopoietic cells through PAK2.

Acute myeloid leukaemia (AML) is an aggressive haematological malignancy characterised by the clonal proliferation of myeloid progenitor cells in the bone marrow and peripheral blood. Dysregulation of the Wnt/{beta}-catenin pathway has been implicated in the establishment and maintenance of leukaemic stem cells in AML, where higher expression of {beta}-catenin promotes clonogenic capacity, drug resistance and inferior survival. The finding that low levels of Wnt signalling are necessary to maintain normal haematopoiesis makes {beta}-catenin an attractive therapeutic target; however, drug design has been hampered by a poor understanding of its molecular interactions in leukaemia cells. To address this, we previously characterised the {beta}-catenin interactome in myeloid cells and identified a plethora of novel interacting proteins. One such interactor was Target of EGR1 (TOE1), a member of the Asp-Glu-Asp-Asp (DEDD) family of deadenylases with previously uncharacterised function in haematopoietic cells. The {beta}-catenin:TOE1 interaction was detected in the nuclear and cytosolic compartments of myeloid cell lines and primary AML samples, and {beta}-catenin depletion was found to promote the cytosolic accumulation of TOE1. Furthermore, TOE1 levels were found to be overexpressed in primary AML blasts versus normal cord-blood derived CD34+ haematopoietic stem and progenitor cells (HSPCs), suggesting that TOE1 levels may be dysregulated in leukaemia. TOE1 depletion abrogated Wnt signalling capacity (TCF/LEF activity), potentially via reduced stability/translatability of the Wnt transcription factor lymphoid enhancing factor 1 (LEF-1). TOE1 depletion further suppressed the proliferation and survival of myeloid leukaemia cell lines (HEL and OCI-AML2) and primary human CD34+ HSPC; however, this could not be fully explained through LEF-1 alone, since OCI-AML2 do not express LEF-1. Using tandem mass tag (TMT)-labelling coupled to mass spectrometry analysis in TOE1 deficient HEL and OCI-AML2 cells, we identified and validated p21 (RAC1) activated kinase 2 (PAK2) as a downregulated target that could reduce the proliferation (but not survival) of AML cell lines. Interestingly, ectopic expression of PAK2 was able to partially rescue the proliferation defect in TOE1 depleted myeloid cell lines and primary human CD34+ HSPC. In summary, these data reveal TOE1 as novel interacting partner for {beta}-catenin in haematological cells capable of modulating Wnt signalling output via LEF-1, and as a novel mediator of growth and survival in human HSPC and AML cells partly through PAK2 regulation.

cancer biology↗

bioRxiv2025

Copper Driven Mutualism of Candida albicans and Staphylococcus aureus Interkingdom Biofilms

Although fungi and bacteria commonly coexist within polymicrobial communities, the molecular mechanisms underlying their interactions are still not well understood. Here, we show that the fungus Candida albicans forms biofilms with the bacterium Staphylococcus aureus along a nutritional axis of mutualism and propose that "a copper economy" shapes fungal-bacterial biofilm interactions. Using in vitro biofilms formed on plastic, we found that dual species biofilms are consistently larger than single-species counterparts, indicating a cooperative interaction. Dual species proteomic analysis revealed non-reciprocal copper handling: C. albicans increased copper uptake via transporter Ctr1, while S. aureus enhanced copper export via regulator CsoR and export chaperone CopZ. Dual species biofilms exhibited specific sensitivity to both copper depletion and supplementation, with corresponding reductions in biomass. We identified fungal copper import as the crucial element in mutualistic interactions between C. albicans and staphylococcal species. Moreover, fungal hyphae served as a critical scaffold for biofilm architecture, a role that was compromised under copper-replete conditions. Notably, copper nanoparticles disrupted these dual species biofilms, highlighting a potential therapeutic avenue. Furthermore, we extend the role of C. albicans copper import to mutualistic interactions with additional bacterial species. Our findings establish copper as a central mediator of C. albicans and S. aureus cooperation and suggest that a "copper economy" underpins mutualistic interactions in biofilms.

microbiology↗

bioRxiv2024

Golgins support extracellular matrix secretion by collectively maintaining the Golgi structure-function relationship

The secretion of extracellular matrix (ECM) proteins is vital to the maintenance of tissue health. One major control point of this process is the Golgi apparatus, whose dysfunction causes numerous connective tissue disorders. Golgi function is tightly linked to its structure, which is maintained by the cytoskeleton and Golgi organising proteins. We sought to investigate the role of two of these organising proteins, the golgins GMAP210 and Golgin-160, in ECM secretion. We found that loss of either protein had distinct impacts on Golgi organisation. GMAP210 loss caused cisternal fragmentation and dilation, alongside the accumulation of tubulovesicular structures. Meanwhile, Golgin-160 knockout lead to Golgi fragmentation and vesicle build-up. Nonetheless, loss of each protein had a similar impact on ECM secretion and glycosaminoglycan synthesis. We therefore propose that golgins are collectively required to create the correct physical-chemical space to support efficient ECM protein secretion and modification. This is the first time that Golgin-160 has been shown to be required for ECM secretion. SummaryIn this study, Thompson et al demonstrate that two cis-Golgi golgins, GMAP210 and Golgin-160, have distinct, non-redundant roles in maintaining Golgi organisation and that both are required to support the efficient secretion, assembly, and modification of extracellular matrix proteins.

cell biology↗

bioRxiv2024

DNAAF9 (Shulin) and ARL3 regulate the transport and activation of ciliary Outer Dynein Arms

Multiciliogenesis requires large-scale biosynthesis of motility-powering axonemal inner and outer dynein arm motors (IDA and ODA) prior to their intraflagellar transport (IFT) into cilia. ODAs are inhibited by the packaging chaperone Shulin during ciliogenesis in T. thermophila. How Shulin is released for ODAs to become active inside cilia remains unclear. We establish interactions between DNAAF9 (human Shulin) and mammalian ODA subunits, IFT proteins and the ciliary small GTPase ARL3 using proteomics and in vitro reconstitutions. Mutagenesis combined with biochemical and structural studies reveal that DNAAF9 and Shulin preferentially bind the active Arl3-GTP state. GTP-loaded Arl3 can access, bind and displace Shulin from the packaged ODA-Shulin complex. We propose that once the inhibited ODA complex enters growing cilia, Arl3-GTP displaces Shulin (DNAAF9) and sequesters it away from ODAs promoting activation of their motility specifically inside cilia.

cell biology↗

bioRxiv2023

Structure of the Commander endosomal trafficking complex linked to X-linked intellectual disability/Ritscher-Schinzel syndrome

The Commander complex is required for endosomal recycling of diverse transmembrane cargos and is mutated in Ritscher-Schinzel syndrome. It comprises two subassemblies; Retriever composed of VPS35L, VPS26C and VPS29, and the CCC complex which contains ten subunits COMMD1-COMMD10 and two coiled-coil domain-containing (CCDC) proteins CCDC22 and CCDC93. Combining X-ray crystallography, electron cryomicroscopy and in silico predictions we have assembled a complete structural model of Commander. Retriever is distantly related to the endosomal Retromer complex but has unique features preventing the shared VPS29 subunit from interacting with Retromer-associated factors. The COMMD proteins form a distinctive hetero-decameric ring stabilised by extensive interactions with CCDC22 and CCDC93. These adopt a coiled-coil structure that connects the CCC and Retriever assemblies and recruits a sixteenth subunit, DENND10, to form the complete Commander complex. The structure allows mapping of disease-causing mutations and reveals the molecular features required for the function of this evolutionarily conserved trafficking machinery.

biochemistry↗

bioRxiv2022

Clinical mutations in WDR34 lead to diverse impacts on the assembly and function of dynein-2

The primary cilium is a sensory organelle, receiving signals from the external environment and relaying them into the cell. Mutations in proteins required for transport in the primary cilium result in ciliopathies, a group of genetic disorders that commonly lead to the malformation of organs such as the kidney, liver and eyes and skeletal dysplasias. Motor proteins dynein-2 and kinesin-2 mediate retrograde and anterograde transport respectively in the cilium. WDR34, a dynein-2 intermediate chain, is required for the maintenance of cilia function. Here, we investigated WDR34 mutations identified in Jeune syndrome, short-rib polydactyly syndrome or asphyxiating thoracic dysplasia patients. There is a poor correlation between genotype and phenotype in these cases making diagnosis and treatment highly complex. We set out to define the biological impacts on cilia formation and function of WDR34 mutations by stably expressing the mutant proteins in WDR34 knockout cells. WDR34 mutations led to different spectrums of phenotypes. Quantitative proteomics demonstrated changes in dynein-2 assembly, whereas initiation and extension of the axoneme, IFT-B protein localization, transition zone (TZ) integrity, and Hedgehog signalling were also affected. Summary statementDisease-associated mutations in WDR34 are found to have diverse impacts on ciliogenesis and cilia function following stable expression in a WDR34 knockout cell model.

cell biology↗

bioRxiv2022

Nutrient and salt depletion synergistically boosts glucose metabolism in individual Escherichia coli cells

The interaction between a cell and its environment shapes fundamental intracellular processes such as cellular metabolism. In most cases growth rate is treated as a proximal metric for understanding the cellular metabolic status. However, changes in growth rate might not reflect metabolic variations in individuals responding to environmental fluctuations. Here we use single-cell microfluidics-microscopy combined with transcriptomics, proteomics and mathematical modelling to quantify the accumulation of glucose within Escherichia coli cells. In contrast to the current consensus, we reveal that environmental conditions which are comparatively unfavourable for growth, where both nutrients and salinity are depleted, increase glucose accumulation rates in individual bacteria and population subsets. We find that these changes in metabolic function are underpinned by variations at the translational and posttranslational level but not at the transcriptional level and are not dictated by changes in cell size. The metabolic response-characteristics identified greatly advance our fundamental understanding of the interactions between bacteria and their environment and have important ramifications when investigating cellular processes where salinity plays an important role.

microbiology↗

bioRxiv2021

MIA3/TANGO1 enables efficient secretion by constraining COPII vesicle budding

Complex machinery is required to drive secretory cargo export from the endoplasmic reticulum, an essential process in eukaryotic cells. In vertebrates, the Mia3 gene encodes two major forms of Transport ANd Golgi Organization Protein 1 (TANGO1S and TANGO1L). Here, using genome engineering of human cells, light microscopy, secretion assays, genomics, and proteomics we show that disruption of the longer form, TANGO1L, results in relatively minor defects in secretory pathway organization and function including limited impacts on procollagen secretion. In contrast, loss of both long and short forms results in major defects in cell organization and secretion. These include a failure to maintain the localization of ERGIC53 and SURF4 to the ER-Golgi Intermediate Compartment and dramatic changes to the ultrastructure of the ER-Golgi interface. Disruption of TANGO1 expression also causes significant changes in early secretory pathway gene and protein expression. Disruption of both TANGO1L and TANGO1S expression impairs secretion not only of large proteins, including procollagens, but of all types of secretory cargo including small soluble proteins. Our data support a general role for Mia3/TANGO1 in maintaining both secretory pathway structure and function in vertebrate cells.

cell biology↗

bioRxiv2020

Novel mechanisms of efflux-mediated levofloxacin resistance and reduced amikacin susceptibility in Stenotrophomonas maltophilia.

Fluoroquinolone resistance in Stenotrophomonas maltophilia is multi-factorial, but the most significant factor is production of efflux pumps, particularly SmeDEF. Here we report that mutations in the glycosyl transferase gene smlt0622 in S. maltophilia K279a mutant K M6 cause constitutive activation of SmeDEF production, leading to elevated levofloxacin MIC. Selection of a levofloxacin-resistant K M6 derivative, K M6 LEVR, allowed identification of a novel two-component regulatory system, Smlt2645/6 (renamed as SmaRS). The sensor kinase Smlt2646 (SmaS) is activated by mutation in K M6 LEVR causing over-production of two novel ABC transporters and the known aminoglycoside efflux pump SmeYZ. Over-production of one ABC transporter, Smlt1651-4 (renamed as SmaCDEF) causes levofloxacin resistance in K M6 LEVR. Over-production of the other ABC transporter, Smlt2642/3 (renamed SmaAB) and SmeYZ both contribute to the elevated amikacin MIC against K M6 LEVR. Accordingly, we have identified two novel ABC transporters associated with antimicrobial drug resistance in S. maltophilia, and two novel regulatory systems whose mutation causes resistance to levofloxacin, clinically important as a promising drug for monotherapy against this highly resistant pathogen.

microbiology↗

bioRxiv2020

Characterisation of the transcriptome and proteome of SARS-CoV-2 using direct RNA sequencing and tandem mass spectrometry reveals evidence for a cell passage induced in-frame deletion in the spike glycoprotein that removes the furin-like cleavage site

Direct RNA sequencing using an Oxford Nanopore MinION characterised the transcriptome of SARS-CoV-2 grown in Vero E6 cells. This cell line is being widely used to propagate the novel coronavirus. The viral transcriptome was analysed using a recently developed ORF-centric pipeline. This revealed the pattern of viral transcripts, (i.e. subgenomic mRNAs), generally fitted the predicted replication and transcription model for coronaviruses. A 24 nt in-frame deletion was detected in subgenomic mRNAs encoding the spike (S) glycoprotein. This feature was identified in over half of the mapped transcripts and was predicted to remove a proposed furin cleavage site from the S glycoprotein. This motif directs cleavage of the S glycoprotein into functional subunits during virus entry or exit. Cleavage of the S glycoprotein can be a barrier to zoonotic coronavirus transmission and affect viral pathogenicity. Allied to this transcriptome analysis, tandem mass spectrometry was used to identify over 500 viral peptides and 44 phosphopeptides, covering almost all of the proteins predicted to be encoded by the SARS-CoV-2 genome, including peptides unique to the deleted variant of the S glycoprotein. Detection of an apparently viable deletion in the furin cleavage site of the S glycoprotein reinforces the point that this and other regions of SARS-CoV-2 proteins may readily mutate. This is of clear significance given the interest in the S glycoprotein as a potential vaccine target and the observation that the furin cleavage site likely contributes strongly to the pathogenesis and zoonosis of this virus. The viral genome sequence should be carefully monitored during the growth of viral stocks for research, animal challenge models and, potentially, in clinical samples. Such variations may result in different levels of virulence, morbidity and mortality.

microbiology↗

bioRxiv2019

A small membrane protein critical to both the offensive and defensive capabilities of Staphylococcus aureus.

Staphylococcus aureus is a major human pathogen, where the widespread emergence of antibiotic resistance is making infections more challenging to treat. Toxin induced tissue damage and resistance to the hosts immune system are well established as critical to its ability to cause disease. However, recent attempts to study S. aureus pathogenicity at a population level have revealed significant complexity and hierarchical levels of regulation. In an effort to better understand this we have identified and characterized a principle effector protein, MasA. The inactivation of this small highly-conserved membrane protein simultaneously disrupts toxin production and impairs S. aureus ability to resist several aspects of the innate immune system. These pleiotropic effects are mediated by both a change in the stability of the bacterial membrane and the dysregulation of iron homeostasis, which results in a significant impairment in the ability of S. aureus to cause infection in both a subcutaneous and a sepsis model of infection. That proteins with such major effects on pathogenicity remain unidentified in a bacterium as well studied as S. aureus demonstrates how incomplete our understanding of their ability to cause disease is, an issue that needs to be addressed if effective control and treatment strategies are to be developed.

microbiology↗

bioRxiv2018

Subtractive Interaction Proteomics Reveal a Network of Signaling Pathways Activated by an Oncogenic Transcription Factor in Acute Myeloid Leukemia

Acute myeloid leukemias (AML) are characterized by recurrent genomic alterations, often in transcriptional regulators, which form the basis on which current prognostication and therapeutic intervention is overlaid. In AML transformation can often be attributed to single chromosomal aberrations encoding oncogenes, such as t(15;17)-PML/RAR or t(6;9)-DEK/CAN but it is unclear how these aberrant transcription factors drive leukemic signaling and influence cellular responses to targeted therapies. Here we show that by using a novel \"subtractive interaction proteomics\" approach, the high risk AML-inducing oncogene t(6;9)-DEK/CAN directly activates signaling pathways that are driven by the ABL1, AKT/mTOR, and SRC family kinases. The interplay of these signaling pathways creates a network with nodes that are credible candidates for combinatorial therapeutic interventions. These results reveal specific interdependencies between nuclear oncogenes and cancer signaling pathways thus providing a foundation for the design of therapeutic strategies to better address the complexity of cancer signaling.\n\nGraphical Abstract\n\nO_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=173 SRC=\"FIGDIR/small/464958_ufig1.gif\" ALT=\"Figure 1\">\nView larger version (28K):\norg.highwire.dtl.DTLVardef@159295corg.highwire.dtl.DTLVardef@aa83d1org.highwire.dtl.DTLVardef@b41e11org.highwire.dtl.DTLVardef@122156e_HPS_FORMAT_FIGEXP M_FIG C_FIG

cancer biology↗

bioRxiv2018

Dynein-2 is required for a functional transition zone and bidirectional ciliary trafficking

The dynein-2 microtubule motor is the retrograde motor for intraflagellar transport. Mutations in dynein-2 components cause skeletal ciliopathies, notably Jeune syndrome. Dynein-2 comprises a heterodimer of two non-identical intermediate chains, WDR34 and WDR60. Here, we use knockout cell lines to demonstrate that each intermediate chain has a distinct role in cilia function. Both proteins are required to maintain a functional transition zone and for efficient bidirectional intraflagellar transport, only WDR34 is essential for axoneme extension. In contrast, only WDR60 is essential for co-assembly of the other subunits. Furthermore, WDR60 cannot compensate for loss of WDR34 or vice versa. This work defines a functional asymmetry to match the subunit asymmetry within the dynein-2 motor. Analysis of causative point mutations in WDR34 and WDR60 can partially restore function to knockout cells. Our data show that Jeune syndrome is caused by defects in transition zone architecture as well as intraflagellar transport.\n\nSUMMARYHere, Vuolo and colleagues use engineered knockout human cell lines to define roles for dynein-2 intermediate chains. WDR34 is required for axoneme extension, while WDR60 is not. Both subunits are required for cilia transition zone organization and bidirectional intraflagellar transport.

cell biology↗