Biology subjects

Humbert, P. O.

Publications and source records attributed to Humbert, P. O..

8 recordsLinked to original sources

bioRxiv2026

Partitioning of nuclear material into apoptotic fragments through establishment of asymmetric cell death morphology

Cellular material in apoptotic cells must be efficiently cleared by phagocytes to maintain tissue homeostasis. Defects in this process can lead to the onset of secondary necrosis and the release of intracellular contents such as damage associated molecular patterns (DAMPs) and autoantigens that are often derived from the nucleus. Therefore, appropriate handling and clearance of apoptotic material is vital to prevent unwanted inflammatory response and the onset of autoimmune disorders. However, how nuclear material is packaged by apoptotic cells for effective clearance by phagocytes is not well understood. By utilising murine models of apoptosis, we observed that a distinct subset of large extracellular vesicles generated from apoptotic thymocytes, known as apoptotic bodies (ApoBDs), can harbour the majority of nuclear contents. Mechanistically, we discovered that apoptotic cells can asymmetrically partition the nucleus into a single large membrane bleb located at one side of the cell, with other cellular contents such as mitochondria and acid organelles distributed to the opposite side. Whilst this newly observed apoptotic morphology, coined as asymmetric cell death morphology (AsyCDM), is morphologically similar to the process of erythroblast enucleation, pharmacological compounds that could interfere with erythroblast enucleation did not block the establishment of AsyCDM during apoptosis. Notably, AsyCDM was reliant on the contractile forces generated by ROCK1-dependent plasma membrane blebbing. Taken together, this study suggests that intracellular contents are partitioned into different ApoBD subsets during apoptosis through a regulated process driven by ROCK1-dependent actomyosin contraction.

cell biology↗

bioRxiv2025

CDK9 interacts with a RanGTP-NEMP1-Importin β complex to regulate erythroid enucleation

Erythroid enucleation is the final stage of erythroid terminal differentiation and involves the separation of an orthochromatic erythroblast into two daughter cells; a pyrenocyte containing the extruded nucleus, and a reticulocyte that will become a red blood cell. Our previous work identified CDK9 as a regulator of erythroid enucleation that appears to act independently of its known role in regulating RNA polymerase II transcription, suggesting the potential for a new CDK9 role. Using a co-immunoprecipitation and mass spectrometry approach, we identified the interactome of CDK9 in differentiating erythroblasts. We show that CDK9 interacts with a RanGTP-NEMP1-Importin-{beta} complex during erythroid terminal differentiation, and inhibition of importin-{beta} in erythroblasts blocks erythroid enucleation. Using imaging analysis and functional assays of enucleating erythroblasts, we show that CDK9 and importin-{beta} co-locate at a critical site of activity opposite to the nucleus before nuclear extrusion and we describe a novel finding that physically links CDK9 and importin-{beta} activity prior to CaM/Ca2+ signalling and subsequent F-actin activity to achieve enucleation. Key PointsO_LIImportin-{beta} physically interacts with CDK9 in erythroid cells and is a novel regulator of erythroid enucleation C_LIO_LICDK9 and importin-{beta} are required upstream of CaM/Ca2+ signalling to enable nuclear extrusion C_LI

cell biology↗

bioRxiv2022

Structural analysis of human papillomavirus E6 interactions with Scribble PDZ domains

The cell polarity regulator Scribble has been shown to be a critical regulator of the establishment and development of tissue architecture, and its dysregulation promotes or suppresses tumor development in a context dependent manner. Scribble activity is subverted by numerous viruses. This includes human papillomaviruses (HPVs), who target Scribble via the E6 protein. Binding of E6 from high-risk HPV strains to Scribble via a C-terminal PDZ binding motif leads to Scribble degradation in vivo. However, the precise molecular basis for Scribble-E6 interactions remains to be defined. We now show that Scribble PDZ1 and PDZ3 are the major interactors of HPV E6 from multiple high-risk strains, with each E6 protein displaying a unique interaction profile. We then determined crystal structures of Scribble PDZ1 and PDZ3 domains in complex with the PBM motifs of E6 from HPV strains 16, 18 and 66. Our findings reveal distinct interaction patterns for each E6 PBM motif from a given HPV strain, suggesting that a complex molecular interplay exists that underpins the overt Scribble-HPV E6 interaction and controls E6 carcinogenic potential.

biochemistry↗

bioRxiv2022

E-cadherin in developing murine T cells controls spindle alignment and differentiation during β-selection

A critical stage of T cell development is {beta}-selection; at this stage the TCR{beta} chain is generated and the developing T cell starts to acquire antigenic specificity. Progression through {beta}-selection is assisted by a low affinity interaction between the nascent TCR{beta} chain and peptide presented on stromal MHC and external cues provided by the niche, including Notch and CXCR4. In this study, we reveal the importance of a new cue within the murine developing T cell niche which is critical for T cell development. E-cadherin mediates cell-cell interactions and influences cell fate in many developmental systems. In developing T cells E-cadherin contributed to the formation of an immunological synapse and the alignment of the mitotic spindle with the polarity axis during division, which facilitated subsequent T cell development. Collectively, these data highlight a new aspect of the developing T cell niche and provide insights into the role of E-cadherin in the {beta}-selection stage of T cell development.

cell biology↗

bioRxiv2022

A Drosophila in vivo chemical screen reveals that combination drug treatment targeting MEK and DGKα mitigates Ras-driven polarity-impaired tumourigenesis.

The RAS oncogene and upregulation of the RAS signalling pathway is highly prevalent in human cancer, and therefore, therapeutically targeting the RAS pathway is a common treatment in cancer. However, RAS pathway upregulation is not sufficient to drive malignant cancer, since senescence mechanisms prevent cancer progression. Thus, additional mutations, such as mutations that prevent senescence or alter the tissue architecture (cell polarity), are required for RAS-driven tumour progression. Moreover, targeting RAS-driven cancers with RAS pathway inhibitors can often lead to undesirable side-effects and to drug resistance. Thus, identifying compounds that synergise with RAS-pathway inhibitors would enable lower doses of the RAS pathway inhibitors to be used and also decrease the acquisition of drug resistance. Here, in a boutique chemical screen using a Drosophila model of Ras-driven cell polarity-impaired cancer, we have identified compounds that reduce tumour burden by synergising with subtherapeutic doses of the RAS pathway inhibitor, Trametinib, which inhibits mitogen-activated kinase kinase (MEK). Analysis of one of the hits from the screen, Ritanserin, which targets serotonin receptors and diacy glycerol kinase alpha (DGK), revealed that DGK was the critical target in its synergism with Trametinib. We show that human mammary epithelial cells harbouring the H-RAS oncogene and knockdown of the cell polarity gene, SCRIB, are also sensitive to treatment with low doses of Trametinib and DGK inhibition. Mechanistically, DGK inhibition synergises with Trametinib by inhibiting MEK and mTOR activity. Altogether, our results provide evidence that targeting RAS-driven human cancers with RAS pathway and DGK inhibitors will be an effective combination therapy.

cancer biology↗

bioRxiv2021

A Scribble-E-cadherin complex controls daughter cell patterning by multiple mechanisms

The fate of the two daughter cells is intimately connected to their positioning, which is in turn regulated by cell junction remodelling and orientation of the mitotic spindle. How multiple cues are integrated to dictate the ultimate patterning of daughters is not clear. Here, we identify novel mechanisms of regulation of daughter positioning in single MCF10A cells. The polarity protein, Scribble, links E-cadherin to NuMA and Arp2/3 signalling for sequential roles in daughter positioning. First Scribble transmits cues from E-cadherin localised in retraction fibres to control orientation of the mitotic spindle. Second, Scribble re-locates to the junction between the two daughters to allow a new E-cadherin-based-interface to form between them, influencing the width of the nascent daughter-daughter junction, generation of filopodia and subsequent cell patterning. Thus, E-cadherin and Scribble dynamically relocate to different intracellular sites during cell division to orient the mitotic spindle and control placement of the daughter cells after cell division.

cell biology↗

bioRxiv2020

Structural basis of the human Scribble-Vangl2 association in health and disease

Scribble is a critical cell polarity regulator that has been shown to work as either an oncogene or tumor suppressor in a context dependent manner, and also impacts cell migration, tissue architecture and immunity. Mutations in Scribble lead to neural tube defects in mice and humans, which has been attributed to a loss of interaction with the planar cell polarity regulator Vangl2. We show that the Scribble PDZ domains 1, 2 and 3 are able to interact with the C-terminal PDZ binding motif of Vangl2 and have now determined crystal structures of these Scribble PDZ domains bound to the Vangl2 peptide. Mapping of mammalian neural tube defect mutations reveal that mutations located distal to the canonical PDZ domain ligand binding groove can not only ablate binding to Vangl2 but also disrupt binding to multiple other signaling regulators. Our findings suggest that PDZ-associated neural tube defect mutations in Scribble may not simply act in a Vangl2 dependent manner but as broad-spectrum loss of function mutants by disrupting the global Scribble-mediated interaction network.

cell biology↗

bioRxiv2018

Structural analysis of phosphorylation-associated interactions of MCC to Scribble PDZ domains

Scribble is a crucial adaptor protein that plays a pivotal role during establishment and control of cell polarity, impacting many physiological processes ranging from cell migration to immunity and organization of tissue architecture. Scribble harbors a leucine-rich repeat domain and four PDZ domains, which mediate most of Scribbles interactions with other proteins. It has become increasingly clear that posttranslational modifications substantially impact Scribble-ligand interactions, with phosphorylation being a major modulator of binding to Scribble. To better understand how Scribble PDZ domains direct cell polarity signalling and how phosphorylation impacts this process, we investigated human Scribble interactions with MCC (mutated in colorectal cancer). We systematically evaluated the ability of all four individual Scribble PDZ domains to bind the PDZ-binding motif (PBM) of MCC as well as MCC phosphorylated at the -1 Ser position. We show that Scribble PDZ1 and PDZ3 are the major interactors with MCC, and that modifications to Ser at the -1 position in the MCC PBM only has a modest effect on binding to Scribble PDZ domains. We then examined the structural basis for these observations by determining the crystal structures of Scribble PDZ1 domain bound to both the unphosphorylated MCC PBM as well as phosphorylated MCC. Our structures indicated that phospho-Ser at the -1 position in MCC is not involved in major contacts with Scribble PDZ1, and in conjunction with our affinity measurements suggest that the impact of phosphorylation at the -1 position of MCC extends beyond a simple modulation of the affinity for Scribble PDZ domains.

biochemistry↗