Biology subjects

Anderson, R. A.

Publications and source records attributed to Anderson, R. A..

15 recordsLinked to original sources

bioRxiv2026

Meiotic kinetochore reconstitution identifies chromosome segregation factors

Kinetochores are complex protein assemblies at centromeres that direct and regulate chromosome segregation1. During gamete formation by meiosis, kinetochore specialisations drive two sequential and distinct segregation events to partition half the genome to the offspring2,3. Age-dependent segregation errors in human oocytes are a major cause of reproductive failure and are linked to kinetochore dysfunction4-6, however the molecular composition and function of mammalian meiotic centromeric chromatin and kinetochores remains poorly defined. Here, we reconstitute vertebrate meiosis I and II centromeric chromatin using meiosis I and II endogenous proteins to uncover conserved regulators of chromosome segregation in mammalian meiosis. We find that, in addition to canonical kinetochore proteins, a specific subset of chromatin regulators is enriched on meiotic centromeric chromatin. We discover previously unrecognized kinetochore proteins conserved from frogs to mouse and humans and demonstrate their localisation at mammalian oocyte centromeres. Among these, we show that the newly identified centromeric protein HSPBAP1, is required to maintain chromosome integrity and ensure faithful segregation in both mouse and human oocytes. These findings define the composition of vertebrate meiotic kinetochores and uncover conserved factors that safeguard mammalian meiotic chromosome segregation, a highly error-prone process and major cause of aneuploidy and reproductive failure in humans7.

cell biology↗

bioRxiv2025

Lipid Transfer Proteins and PI4KIIα Generate a Phosphoinositide-Linked Proteome

Phosphoinositide (PIPn) lipid second messengers in membranes regulate a myriad of cellular processes. In the cytosol, the phosphatidylinositol (PI) 3-kinase (PI3K)/Akt pathway is scaffolded on IQGAP1 to facilitate the activation of Akt by the synthesis of PI3,4,5P3. In the nucleus, PIPn signaling occurs in regions devoid of membranes via their stable association with proteins. While several of these proteins have been identified, understanding the extent and impact of protein-linked PIPn signaling warrants further investigation. The tumor suppressor p53, was shown in the companion paper to be regulated by PI transfer proteins (PITPs) and a PI 4-kinase (PI4KII), which are required to form p53-PIPn complexes that assemble a nuclear PI3K/Akt pathway. Here we report that class I PITPs (PITP/{beta}) and PI 4-kinase initiate PIPn linkages to many different proteins. PITP/{beta} and PI4KII accumulate in the nucleoplasm in response to stress and are necessary to synthesize nuclear PIPns linked to proteins. These PITP/{beta}-dependent protein-PIPn complexes are detected by metabolically labeling cells with the PIPn precursor [H3]-myo-inositol and resist denaturation and SDS-PAGE, indicating that these protein-PIPn complexes represent a putative posttranslational modification. Proteomic analyses of proteins that are regulated by PITP/{beta} and/or are linked to PI4,5P2 have identified an emerging PIPylome that is enriched in metabolic, signaling, cytoskeletal and DNA repair pathway components. Taken together, these data provide evidence for an emerging proteome with linked PIPns that represent a PIPn signaling paradigm that is distinct from the membrane-localized pathway but utilizes many of the same PIP kinases and phosphatases. In briefPhosphatidylinositol transfer proteins and PI 4-kinase initiate a PIPn-linked protein network in membrane-free regions.

cell biology↗

bioRxiv2025

Multi-nucleation in two-cell human embryos stems from spindle and metaphase plate incoherence in the first mitosis

The first embryonic division in humans is highly error-prone and a source of aneuploidies. Multi-nucleation is prevalent in two-cell human embryos, with unknown cause. Here, we live-image human zygotes to elucidate the features of the first mitosis that predispose embryos to multinucleation. We show that failure to collect chromosomes into a single mass and establish a bipolar spindle during zygotic metaphase leads to severe multi-nucleation. We find that KIF10/CENP-E kinesin activity is essential to prevent the formation of multiple spindle poles and to congress chromosomes onto a metaphase plate. Furthermore, although the spindle assembly checkpoint mediates a delay in response to a highly disorganised spindle, KIF10/CENP-E-inhibited embryos ultimately undergo the first mitosis with a chaotic anaphase. Therefore, defective chromosome congression in the zygote combined with a failure to sense this error cause multi-nucleation. Remarkably, multi-nucleation can be corrected during the second mitotic division in a KIF10/CENP-E-dependent manner. We suggest that multi-nucleation may be a safeguarding mechanism to prevent chromosome loss during the highly error-prone first embryonic mitosis.

cell biology↗

bioRxiv2025

Chromosome Ageing Occurs at the Primordial Follicle Stage in both Mouse and Human Oocytes

Inherited aneuploidy is one of the most common causes of human genetic disease affecting approximately 20% of all human embryos and is the major cause of non-conception in older women. Inherited aneuploidies typically arise from meiotic chromosome segregation errors and are strongly affected by maternal age. Cohesin, a protein complex that mediates cohesion between sister chromatids, becomes depleted from oocyte chromosomes with age in mice, potentially contributing to the high rate of aneuploidy in older mouse oocytes. However, it remains unclear at which stage of oogenesis ageing affects chromatid cohesion in mice and whether ageing similarly affects cohesin in human oocytes. Here we use fluorescence in situ hybridisation to assess at which stages of oogenesis ageing alters chromatid separation in mice and in humans. We show that chromatid separation increases with age while oocytes remain dormant in primordial follicles in both mice and humans and that this effect of age on chromatid separation is detectable in fertile women of child-bearing age. Furthermore, we show that this age-dependent increase in chromatid separation is accompanied by age-dependent depletion of the acetylated SMC3-marked cohesive subpopulation of cohesin from oocytes at the dormant primordial follicle stage in both mice and humans. These data suggest that ageing impacts on oocyte chromosomes while they remain dormant in primordial follicles, and that this aspect of oocyte chromosome ageing is shared in both mice and humans. Improving our understanding of these pathways may allow strategies to slow or prevent this aspect of oocyte ageing to be developed.

developmental biology↗

bioRxiv2025

Regulation of the MDM2-p53 Nexus by a Nuclear Phosphoinositide and Small Heat Shock Protein Complex

The tumor suppressor p53 maintains genome stability in the setting of cellular stress and is frequently mutated in cancer. The stability of p53 is regulated by its interaction with the oncoprotein MDM2, a ubiquitin E3 ligase. Recently, nuclear phosphoinositides were reported to bind and stabilize p53. Here, we report that genotoxic stress induces the type I phosphatidylinositol phosphate kinase (PIPKI) and its product phosphatidylinositol 4,5-bisphosphate (PIP2) to bind and regulate the stability and function of MDM2. Following genotoxic stress, nuclear PIPKI binds to MDM2 to generate a complex of MDM2 and PIP2. PIP2 binding to MDM2 differentially regulates the recruitment of the small heat shock proteins (sHSPs) B-crystallin (BC) and HSP27 to the MDM2-PIP2 complex, acting as an on-off switch that regulates MDM2 stability, downstream targets, ubiquitination activity, and interaction with p53. Our results demonstrate an unexpected role for nuclear phosphoinositides conferring specificity to the MDM2-PIP2-sHSPs association. Notably, the differential engagement of BC and HSP27 reveals that sHSPs are not merely passive chaperones but play active, selective roles in fine-tuning MDM2 function and MDM2-p53 nexus. These findings provide a novel therapeutic strategy for targeting this pathway in cancer.

cancer biology↗

bioRxiv2024

Regulation of the poly(A) Polymerase Star-PAP by a Nuclear Phosphoinositide Signalosome

Star-PAP is a noncanonical poly(A) polymerase that controls gene expression. Star-PAP was previously reported to bind the phosphatidylinositol 4-phosphate 5-kinase PIPKI and its product phosphatidylinositol 4,5-bisphosphate, which regulate Star-PAP poly(A) polymerase activity and expression of specific genes. Recent studies have revealed a nuclear PI signaling pathway in which the PI transfer proteins PITP/{beta}, PI kinases and phosphatases bind p53 to sequentially modify protein-linked phosphatidylinositol phosphates and regulate its function. Here we demonstrate that multiple phosphoinositides, including phosphatidylinositol 4-monophosphate and phosphatidylinositol 3,4,5-trisphosphate are also coupled to Star-PAP in response to stress. This is initiated by PITP/{beta} binding to Star-PAP, while the Star-PAP-linked phosphoinositides are modified by PI4KII, PIPKI, IPMK, and PTEN recruited to Star- PAP. The phosphoinositide coupling enhances the association of the small heat shock proteins HSP27/B-crystallin with Star-PAP. Knockdown of the PITPs, kinases, or HSP27 reduce the expression of Star-PAP targets. Our results demonstrate that the PITPs generate Star-PAP-PIPn complexes that are then modified by PI kinases/phosphatases and small heat shock proteins that regulate the linked phosphoinositide phosphorylation and Star-PAP activity in response to stress.

cell biology↗

bioRxiv2024

Enhanced PI3K/Akt Signaling in Response to p85α Loss is Regulated by Endosomal PI3Kα and PI3P

PI3K is a heterodimer of p110 catalytic subunit and p85 adaptor subunit that is activated by agonist-stimulated receptor tyrosine kinases. Although the interaction of p85 with activated receptors recruits p110 to membranes, studies have demonstrated that p85 loss, which occurs commonly in cancer, paradoxically promotes agonist-stimulated PI3K/Akt signaling. We recently demonstrated that p110 localizes to microtubules via MAP4, facilitating its interaction with activated receptor kinases in endosomes to initiate PI3K/Akt signaling. Here, we demonstrate that in response to agonist stimulation and p85 knock down the residual p110, coupled predominantly to p85{beta}, exhibits enhanced microtubule localization, MAP4 binding and interaction with endosomal receptor tyrosine kinases, thereby augmenting PI3K/Akt signaling. The interaction of the C2 domain of p110 with PI3P is required for recruiting p110 into endomembranes and enhancing PI3K/Akt signaling. These findings provide a mechanism for the augmented agonist-stimulated PI3K/Akt signaling upon p85 loss and point to novel therapeutic targets for cancer. IN BRIEFThis study provides the comprehensive mechanism for p85 loss induced and receptor tyrosine kinase stimulated PI3K/Akt signaling.

cell biology↗

bioRxiv2023

Heme biosynthesis regulates BCAA catabolism and thermogenesis in brown adipose tissue

With age, people tend to accumulate body fat and reduce energy expenditure1. Brown (BAT) and beige adipose tissue dissipate heat and increase energy expenditure via the activity of the uncoupling protein UCP1 and other thermogenic futile cycles2,3. The activity of brown and beige depots inversely correlates with BMI and age4-11, suggesting that promoting thermogenesis may be an effective approach for combating age-related metabolic disease12-15. Heme is an enzyme cofactor and signaling molecule that we recently showed to regulate BAT function16. Here, we show that heme biosynthesis is the primary contributor to intracellular heme levels in brown adipocytes. Inhibition of heme biosynthesis leads to mitochondrial dysfunction and reduction in UCP1. Although supplementing heme can restore mitochondrial function in heme-synthesis-deficient cells, the downregulation of UCP1 persists due to the accumulation of the heme precursors, particularly propionyl-CoA, which is a product of branched-chain amino acids (BCAA) catabolism. Cold exposure promotes BCAA uptake in BAT, and defects in BCAA catabolism in this tissue hinder thermogenesis17. However, BCAAs contribution to the TCA cycle in BAT and WAT never exceeds 2% of total TCA flux18. Our work offers a way to integrate current literature by describing heme biosynthesis as an important metabolic sink for BCAAs.

cell biology↗

bioRxiv2023

Regulation of NRF2 by Phosphoinositides and Small Heat Shock Proteins

Reactive oxygen species (ROS) are generated by aerobic metabolism, and their deleterious effects are buffered by the cellular antioxidant response, which prevents oxidative stress. The nuclear factor erythroid 2-related factor 2 (NRF2) is a master transcriptional regulator of the antioxidant response. Basal levels of NRF2 are kept low by ubiquitin-dependent degradation of NRF2 by E3 ligases, including the Kelch-like ECH-associated protein 1 (KEAP1). Here, we show that the stability and function of NRF2 is regulated by the type I phosphatidylinositol phosphate kinase {gamma} (PIPKI{gamma}), which binds NRF2 and transfers its product phosphatidylinositol 4,5-bisphosphate (PtdIns(4,5)P2) to NRF2. PtdIns(4,5)P2 binding recruits the small heat shock protein HSP27 to the complex. Silencing PIPKI{gamma} or HSP27 destabilizes NRF2, reduces expression of its target gene HO-1, and sensitizes cells to oxidative stress. These data demonstrate an unexpected role of phosphoinositides and HSP27 in regulating NRF2 and point to PIPKI{gamma} and HSP27 as drug targets to destabilize NRF2 in cancer. In briefPhosphoinositides are coupled to NRF2 by PIPKI{gamma}, and HSP27 is recruited and stabilizes NRF2, promoting stress-resistance.

cancer biology↗

bioRxiv2023

Lipid transfer proteins initiate nuclear phosphoinositide signaling

Phosphoinositide (PIPn) messengers are present in non-membranous regions of nuclei where they are assembled into a phosphatidylinositol (PI) 3-kinase (PI3K)/Akt pathway that is distinct from the cytosolic membrane-localized pathway. In the nuclear pathway, PI kinases/phosphatases bind the p53 tumor suppressor protein (wild-type and mutant) to generate p53-PIPn complexes (p53-PIPn signalosome) that activate Akt by a PI3,4,5P3-dependent mechanism in non-membranous regions of the nucleus. This pathway is dependent on a source of nuclear PIPns that is poorly characterized. Here we report that a subset of PI transfer proteins (PITPs), which transport PI between membranes to enable membrane-localized PIPn synthesis, also interact with p53 in the nucleus upon genotoxic stress. Class I PITPs (PITP/{beta}) specifically supply the PI required for the generation of p53-PIPn complexes and subsequent signaling in the nucleus. Additionally, the PI 4-kinase PI4KII binds to p53 and the PITPs to catalyze the formation of p53-PI4P. p53-PI4P is then sequentially phosphorylated to synthesize p53-PIPn complexes that regulate p53 stability, nuclear Akt activation and genotoxic stress resistance. In this way, PITP/{beta} and PI4KII bind p53 and collaborate to initiate p53-PIPn signaling by mechanisms that require PI transfer by PITP/{beta} and the catalytic activity of PI4KII. Moreover, the identification of these critical upstream regulators of p53-PIPn signaling point to PITP/{beta} and PI4KII as potential therapeutic targets in this pathway for diseases like cancer. In BreifPhosphatidylinositol transfer proteins and a PI 4-kinase initiate nuclear p53-PIPn signaling in membrane-free regions.

cell biology↗

bioRxiv2023

Age-dependent loss of cohesion protection in human oocytes

Aneuploid human eggs (oocytes) are a major cause of infertility, miscarriage and chromosomal disorders. Such aneuploidies increase greatly as women age, originating from defective linkages between sister-chromatids (cohesion) in meiosis. We found evidence that loss of a specific pool of the cohesin protector protein, shugoshin 2 (Sgo2) contributes to this phenomenon. Our data indicate that Sgo2 preserves sister chromatid cohesion in meiosis by protecting a cohesin bridge between sister chromatids. In human oocytes, Sgo2 localizes to both sub-centromere cups and the pericentromeric bridge which spans the sister chromatid junction. Sgo2 normally colocalizes with cohesin, however, in oocytes from older women, Sgo2 is frequently lost specifically from the pericentromeric bridge and sister chromatid cohesion is weakened. Mps1 and Bub1 kinase activities maintain Sgo2 at sub-centromeres and the pericentromeric bridge. Removal of Sgo2 throughout meiosis I by Mps1 inhibition reduces cohesion protection, increasing the incidence of single chromatids at meiosis II. Therefore, Sgo2 deficiency in human oocytes can exacerbate the effects of maternal age by rendering residual cohesin at pericentromeres vulnerable to loss in anaphase I. Our data show that maternal age-dependent loss of Sgo2 at the pericentromere bridge in human oocytes impairs cohesion integrity and contributes to the increased incidence of aneuploidy observed in human oocytes with advanced maternal age.

cell biology↗

bioRxiv2021

A p53-Phosphoinositide Signalosome Regulates Nuclear Akt Activation

The tumor suppressor p53 and the phosphoinositide 3-kinase (PI3K)-Akt pathway have fundamental roles in regulating cell growth, apoptosis and are frequently mutated in cancer. Here, we show that genotoxic stress induces nuclear Akt activation by a p53-dependent mechanism that is independent from the canonical membrane-localized PI3K-Akt pathway. Upon genotoxic stress a nuclear p53-PI3,4,5P3 complex is generated in regions devoid of membranes by a nuclear PI3K, and this complex recruits all the kinases required to activate Akt and phosphorylate FOXOs, inhibiting DNA damage-induced apoptosis. Wild-type p53 activates nuclear Akt in an on/off fashion upon stress, whereas mutant p53 stimulates high basal Akt activity, indicating a fundamental difference. The nuclear p53-phosphoinositide signalosome is distinct from the canonical membrane-localized pathway and insensitive to PI3K inhibitors currently in the clinic, underscoring its therapeutic relevance. In briefp53 assembles a PI3K-Akt pathway that regulates nuclear Akt activation independent of the canonical pathway on membranes.

cell biology↗

bioRxiv2020

Histone H3.3 Hira chaperone complex contributes to zygote formation in mice and humans.

Elucidating the underlining mechanisms underpinning successful fertilisation is imperative in optimising IVF treatments, and may lead to a specific diagnosis and therefore potential treatment for some infertile couples. One of the critical steps involves paternal chromatin reprogramming, in which compacted sperm chromatin packed by protamines is removed by oocyte factors and new histones, including histone H3.3, are incorporated. This step is critical for the formation of the male pronucleus, without which the zygote contains only 1 pronucleus (1PN), in contrast to normally fertilised zygotes with two-pronuclei (2PN). 1PN zygotes are a frequently observed phenomenon in IVF treatments, therefore aberrant mechanism of action controlling paternal chromatin repackaging may be an important cause of abnormal fertilisation. Hira is the main H3.3 chaperone that governs this protamine-to-histone exchange. In this study, we investigated the maternal functions of two other molecules of the Hira complex, Cabin1 and Ubn1 in the mouse. Loss-of-function Cabin1 and Ubn1 mouse models were developed: their zygotes displayed abnormal 1PN zygote phenotypes, similar to the phenotype of Hira mutants. We then studied human 1PN zygotes, and found that the Hira complex was absent in 1PN zygotes which were lacking the male pronucleus. This result confirms that the role of the Hira complex in male pronucleus formation has coherence from mice to humans. Furthermore, rescue experiments showed that the abnormal 1PN phenotype derived from Hira mutants could be resolved by overexpression of Hira in the mouse oocytes. In summary, we have provided evidence of the role of Hira complex in regulating male pronucleus formation in both mice and humans, that both Cabin1 and Ubn1 components of the Hira complex are equally essential for male pronucleus formation, and that this can be rescued. We present a proof-of-concept experiment that could potentially lead to a personalised IVF therapy for oocyte defects.

developmental biology↗

bioRxiv2018

The Specificity of IQGAP1 Toward the PI3K-Akt Pathway is Dependent on the IQ3 motif

Epidermal growth factor receptor (EGFR) and its downstream phosphatidylinositol 3-kinase (PI3K) pathway are commonly deregulated in many cancers including head and neck cancer (HNC). Recently, we have shown that the IQ motif-containing GTPase-activating protein 1 (IQGAP1) provides a molecular platform to scaffold all the components from the PI3K-Akt pathway and results in the sequential generation of phosphatidylinositol-3,4,5-triphosphate (PI3,4,5P3). This makes the IQGAP1-PI3K scaffold a promising therapeutic target. In addition to the PI3K-Akt pathway, IQGAP1 also scaffolds the Ras-ERK pathway. To identify an IQGAP1 mutant that specifically loses IQGAP1-PI3K signaling but not other functions, we have focused on the IQ3 motif since this region binds with both the PIPK1 and PI3K enzymes and a short peptide derived from this sequence blocks binding and PI3K signaling. An IQ3 deletion mutant ({Delta}IQ3) in IQGAP1 was functionally compared with wild-type (WT) IQGAP1. We found that the IQ3 domain specifically mediates the PI3K-Akt pathway but does not regulate the Ras-ERK pathway. The IQ3 deletion mutant lost interactions with PI3K-Akt components but retained its binding of ERK pathway components and cell surface receptors, EGFR and integrins. In addition, the IQ3 deletion mutant lost regulation of cell migration. Consistently, the IQ3 motif derived peptide blocked Akt activation and led to the blockage of invasion mediated by EGFR organized into an integrin complex by syndecan-4. Taken together, this work has defined the IQGAP1 IQ3 motif as a specific target sequence for the scaffolding of the PI3K-AKT pathway.

cell biology↗

bioRxiv2018

MST1R/RON and EGFR in a complex with syndecans sustain carcinoma S-phase progression by preventing p38MAPK activation

Syndecan-4 (Sdc4) organizes a complex of receptors consisting of its homologue, Sdc2, the receptor tyrosine kinases EGFR and MST1R/RON, and the laminin-binding 3{beta}1 and 6{beta}4 integrins that depends on a docking site within its extracellular domain. A peptide mimetic of the extracellular docking site, synstatin-EGFR (SSTNEGFR), disrupts the receptor complex and prevents the invasion of non-transformed or carcinoma cells that relies on active EGFR. However, the peptide also prevents DNA replication that relies on active MST1R/RON and c-Abl kinase within the complex, resulting in rapid S-phase arrest of head & neck (HN) and breast carcinoma cells. SSTNEGFR does not affect DNA replication in non-transformed oral or breast epithelial cells, but it does block their EGF-dependent invasion. Although EGFR is required as a component of the complex, its kinase activity is not required to sustain S-phase progression in the carcinoma cells, perhaps explaining why many HN and breast carcinomas that overexpress EGFR are nonetheless refractory to EGFR inhibitors. The syndecan-organized receptor complex (Sdc:RTK:ITG complex) appears to suppress stress signals that would otherwise disrupt the replisome engaged in DNA synthesis. SSTNEGFR-treatment of carcinoma cells, or normal oral epithelial cells expressing stress-inducing HPV oncogenes, causes rapid activation of the p38 stress MAPK leading to loss of PCNA from the chromatin and cessation of DNA synthesis. This arrest is independent of the common DNA damage response (DDR) known to activate an S-phase checkpoint, revealing a novel arrest mechanism and a novel receptor complex that is activated on tumor cells to suppress stress-induced proliferation arrest.

cancer biology↗