Biology subjects

Elowe, S.

Publications and source records attributed to Elowe, S..

10 recordsLinked to original sources

bioRxiv2026

ZNF768 regulates expression of E2F1 protein to drive G0/G1 transition and cell cycle progression.

Accurate and tightly coordinated cell cycle progression and cell proliferation are critical for development, growth and homeostasis of an organism. Recently, Zinc finger protein 768 (ZNF768) was identified as a transcription factor driving cellular proliferation, in both a p53-dependent and independent manner. ZNF768 interacts with and represses p53 functions to limit cell cycle delay. Independently, ZNF768 promotes the transcription of key regulators of the cell cycle machinery, although the mechanisms through which this occurs remain unknown. Here, we report that ZNF768 protein levels are tightly regulated during the cell cycle, and its depletion leads to cell cycle exit and induction of quiescence. We found that ZNF768 modulates the cell cycle, at least in part, by controlling expression of the major pro-proliferative transcription factor E2F1 independently of p53 activation. Consequently, depletion of ZNF768, which also represses expression of the key mitotic transcription factor and E2F1 target FOXM1, leads to numerous mitotic errors. Supporting these findings, cancer genomics analyses reveal that ZNF768 expression levels are positively associated with E2F1 and FOXM1 expression levels in human tumors, suggesting that cancer cells might use ZNF768 to override cell cycle arrest, sustain proliferation, and promote cancer progression. Altogether, our results reveal that ZNF768 modulates cell cycle entry and proliferation, at least in part by regulating E2F1 expression.

cell biology↗

bioRxiv2024

A characterization of mitotic and centrosomal defects in a continuum model of Breast Cancer

Errors in mitosis can contribute to aneuploidy and CIN and play a pivotal role in cancer. So the identification of altered mitotic regulators can contribute to the understanding of the development and progression of breast cancer. In the present study we used an in vitro model of disease progression (the MCF10A series of BC continuum) and analyzed the errors of chromosome segregation that occur during the progression of the disease. Our findings indicated that the MCF10A series exhibited several abnormalities in chromosome segregation and its frequency increased with the disease progression. These errors included anaphase lagging chromosomes, micronuclei, nuclear buds, nucleoplasmic bridges, errors of chromosome alignment, and centrosome loss/amplification. Moreover, the presence of centrosome amplification disrupted the proper orientation of the mitotic spindle, resulting in the generation asymmetrical cell lines and aneuploidy in the MCF10A series. Hyper stable kinetochore-microtubule (kt-MT) attachment was also found in premalignant, preinvasive, and invasive cell lines, which can also explain the presence of errors of chromosome alignment. The human transcriptome array also determined possible negative regulators of ciliogenesis that can explain the mechanism of chromosome missegregation that lead to CIN found in the MCF10A series. Collectively, these findings highlight the importance of mitotic defects in the progression of breast cancer.

cell biology↗

bioRxiv2024

EPHA1 and EPHB4 tyrosine kinase receptors regulate epithelial morphogenesis

Organ formation and homeostasis require the coordination of cell-cell adhesion, epithelial cell polarity and orientation of cell division to organize epithelial tissue architecture. We have previously identified proximity protein networks acting downstream of members of the EPH family of tyrosine kinase receptors and found within these networks an enrichment of components associated with cell morphogenesis and cell-cell junctions. Here, we show that two EPH receptors, EPHA1 and EPHB4, are localized to the basolateral domain of Caco-2 cells in spheroidal cultures. Depletion of either EPHA1 or EPHB4 disrupts spheroid morphogenesis, without affecting cell polarity, but via randomizing mitotic spindle orientation during cell division. Strikingly, EPHA1 and EPHB4 exert this function independently of their catalytic activity but still requiring EFN ligand binding. Consistent with this, the most abundantly expressed EPHB4 ligand in Caco-2 cells, EFNB2, is also compartmentalized at the basolateral domain in spheroids, and is required for epithelial morphogenesis. Taken together, our data reveal a new role for EPHRs in epithelial morphogenesis.

cell biology↗

bioRxiv2024

ARHGEF17/TEM4 regulates the cell cycle through control of G1 progression

The Ras homolog (Rho) small GTPases, via their role in regulating the actin cytoskeleton, coordinate diverse cellular functions including cell morphology, adhesion and motility, as well as cell cycle progression, survival and apoptosis. The upstream Rho regulators for many of these functions are unknown. ARHGEF17 (also known as TEM4) is a Rho family guanine nucleotide exchange factor (GEF) that been implicated in cell migration, cell-cell junction formation and the mitotic checkpoint. In this study we characterize the regulation of the cell cycle by TEM4. We demonstrate that TEM4 depleted cells exhibit multiple defects in mitotic entry and duration, spindle morphology, and spindle orientation. In addition, we find that TEM4 insufficiency leads to excessive cortical actin polymerization and cell rounding defects. Mechanistically, we demonstrate that TEM4 depleted cells delay in G1 as a consequence of elevated levels of the G1/S inhibitor p21waf1/cip1 and that TEM4 depleted cells that progress through to mitosis, do so with decreased transcription of CCNB1 and thus attenuated levels of cyclin B. Importantly, cyclin B overexpression in TEM4-depleted cells largely rescues mitotic progression and chromosome segregation defects in anaphase. Our study thus illustrates the consequences of Rho signalling imbalance on cell cycle progression and identifies TEM4 as the first GEF governing Rho GTPase-mediated regulation of G1/S.

cell biology↗

bioRxiv2023

The substrate quality of CK2 target sites has a determinant role on their function and evolution

Most biological processes are regulated by peptide-recognition modules (PRMs) that bind to short linear motifs (SLiMs). Such interactions are rapidly reversible and often occur at low affinity. The protein kinase domain represents one such binding module, and known substrates may have full or only partial matches to the kinase recognition motif, a property known as substrate quality. However, it is not yet clear whether differences in substrate quality represent neutral variation along the phosphosite sequence or if these differences have functional consequences that are subject to selection. We explore this question in detail for the acidophilic kinase CK2. CK2 is well-characterised, clinically important, and a fundamental enzyme for many aspects of cell biology. We show that optimal CK2 sites are phosphorylated at maximal stoichiometries and found in many conditions whereas minimal substrates are phosphorylated at lower stoichiometries, are more dynamic during the cell cycle, and have regulatory functions. Optimal CK2 sites also tend to be older and more conserved than minimal sites, and evolutionary simulations indicate that the substrate quality of CK2 phosphosites is often tuned by selection. For intermediate target sites, increases or decreases to substrate quality may be deleterious, which we demonstrate experimentally for a CK2 substrate at the kinetochore. The results together suggest that minimal and optimal phosphosites are strongly differentiated in terms of their functional and evolutionary properties.

evolutionary biology↗

bioRxiv2022

A Commercial ARHGEF17/TEM4 antibody cross-reacts with Nuclear Mitotic Apparatus protein 1 (NuMA)

The Rho family Guanine nucleotide exchange factor (GEF) ARHGEF17 (also known as TEM4) is a large protein with only 3 annotated regions: an N-terminal actin-binding domain, a Rho-specific dbl homology (DH)- pleckstrin homology (PH) type GEF domain and a seven bladed {beta} propeller fold at the C-terminus with unknown function. TEM4 has been implicated in numerous activities that rely on regulation of the cytoskeleton including cell migration, cell-cell junction formation and the spindle assembly checkpoint during mitosis. Here we have assessed the specificity of a TEM4 polyclonal antibody that has been commonly used as a Western blotting and immunocytochemistry probe for TEM4 in mammalian cells. We find that this antibody, in addition to its intended target, cross-reacts with the Nuclear Mitotic Apparatus Protein 1 (NuMA) in Western blotting and immunoprecipitation, and detects NuMA preferentially in immunocytochemistry. This cross-reactivity, with an abundant chromatin- and mitotic spindle-associated factor, is likely to affect the interpretation of experiments that make use of this antibody probe, in particular by immunocytochemistry and immunoprecipitation.

cell biology↗

bioRxiv2020

Proximity proteomics reveal EPH tyrosine kinase receptors' regulation of cell polarity and identify Par3 scaffold protein as a novel effector of EPH signaling

The EPH family is the largest among receptor tyrosine kinases (RTKs) in humans. In contrast to other RTKs, EPH receptors (EPHRs) cognate ligands, ephrins, are tethered to the cell surface. This results in EPHR-ephrin signaling being mainly involved in short-range cell-cell communication events that regulate cell adhesion, migration and tissue boundary formation. Although EPHRs functions have been broadly studied, the molecular mechanisms by which they control these processes are far from being understood. To address this, we sought to identify new effector proteins acting downstream of EPHRs and determine their role in EPHR-regulated functions. To unravel EPHR-associated signaling complexes under native conditions, we applied a mass spectrometry-based approach, namely BioID proximity labeling. We obtained a composite proximity network from EPHA4, -B2, -B3 and -B4 receptors that comprises 395 proteins, most of which were not previously linked to EPH signaling. A gene ontology and pathway term analysis of the most common candidates highlighted cell polarity as a novel function associated with EPHR activity. We found that EPHA1 and EPHB4 expression is restricted to the basal and lateral membrane domains in polarized Caco-2 3D spheroidal cell cultures. We further discovered that their depletion impairs the compartmentalized distribution of polarity proteins as well as overall spheroid morphogenesis. Moreover, we examined the contribution of a number of candidates, selected from EPHR proximity networks, via loss-of-function in an EPHR-dependent cell segregation assay. We found that depletion of the signaling scaffold PAR-3 blocks cell sorting. We also delineated a signalling complex involving the C-terminal SRC kinase (CSK), whose recruitment to PAR-3 complexes is dependent on EPHR signals. Our work sheds a new light on EPHR signaling networks and describes conceptually novel the mechanisms by which EPHRs signal at the membrane to contribute to the regulation of cellular phenotypes.

molecular biology↗

bioRxiv2020

An unbiased, quantitative and versatile method for determining misaligned and lagging chromosome during mitosis

Accurate chromosome alignment at metaphase facilitates the equal segregation of sister chromatids to each of the nascent daughter cells. Lack of proper metaphase alignment is an indicator of defective chromosome congression and aberrant kinetochore-microtubule attachments which in turn promotes chromosome missegregation and aneuploidy, hallmarks of cancer. Therefore, tools to sensitively and quantitatively measure chromosome alignment at metaphase will facilitate understanding of how changes in the composition and regulation of the microtubule attachment machinery impinge on this process. In this work, we have developed and validated a method based on analytical geometry to measure several indicators of chromosome misalignment. We generated semi-automated and flexible ImageJ2/Fiji pipelines to quantify kinetochore misalignment at metaphase plates as well as lagging chromosomes at anaphase. These tools will ultimately allow sensitive, unbiased, and systematic quantitation of these chromosome segregation defects in cells undergoing mitosis.

cell biology↗

bioRxiv2019

The BUBR1 pseudokinase domain promotes efficient kinetochore PP2A-B56 recruitment to regulate spindle checkpoint silencing and chromosome alignment

The balance of phospho-signalling at outer-kinetochores during mitosis is critical for the accurate attachments between kinetochores and the mitotic spindle and timely exit from mitosis. In humans, a major player in determining this balance is the PP2A-B56 phosphatase which is recruited to the Kinase Attachment Regulatory Domain (KARD) of the Spindle Assembly Checkpoint protein Budding Uninhibited by Benzimidazole 1-related 1 (BUBR1) in a phospho-dependent manner. This event unleashes a rapid, switch-like phosphatase relay that reverses phosphorylation at the kinetochore, extinguishing the checkpoint and promoting anaphase entry. Here, we conclusively demonstrate that the pseudokinase domain of human BUBR1 lacks phosphotransfer activity and that it was maintained in vertebrates because it allosterically promotes KARD phosphorylation. Mutation or removal of this domain results in decreased PP2A-B56 recruitment to the outer kinetochore, attenuated checkpoint silencing and errors in chromosome alignment as a result of imbalance in Aurora B activity. We demonstrate that the functions of the BUBR1 pseudokinase and the BUB1 kinase domains are intertwined, providing an explanation for retention of the pseudokinase domain in certain eukaryotes.

cell biology↗

bioRxiv2018

A type 2 diabetes disease module with a high collective influence for Cdk2 and PTPLAD1 is localized in endosomes

Despite the identification of many susceptibility genes our knowledge of the underlying mechanisms responsible for complex disease remains limited. Here, we identified a type 2 diabetes disease module in endosomes, and validate it for functional relevance on selected nodes. Using hepatic Golgi/endosomes fractions, we established a proteome of insulin receptor-containing endosomes that allowed the study of physical protein interaction networks on a type 2 diabetes background. The resulting collated network is formed by 313 nodes and 1147 edges with a topology organized around a few major hubs with Cdk2 displaying the highest collective influence. Overall, 88% of the nodes are associated with the type 2 diabetes genetic risk, including 101 new candidates. The Type 2 diabetes module is enriched with cytoskeleton and luminal acidification -dependent processes that are shared with secretion-related mechanisms. We identified new signaling pathways driven by Cdk2 and PTPLAD1 whose expression regulate the association of the insulin receptor with TUBA, TUBB, the actin component ACTB and the endosomal sorting markers Rab5c and Rab11a. Therefore, the interactome of internalized insulin receptors reveals the presence of a type 2 diabetes disease module enriched in new layers of feedback loops required for insulin signaling, clearance and islet biology.\n\nAuthor SummaryAccording to the local hypothesis each complex disease can be linked to a well-defined network called the disease module. A disease module can be defined by the topological properties of protein interaction networks. Given the complexity of the whole interaction map the existence of such disease modules remains largely to be tested. Here, we found a type 2 diabetes disease module in insulin receptor-containing endosomes. The disease module contains new pathways that are associated with both insulin signaling, clearance and secretion. Its co-functionality with islets biology may provide a mechanistic rationale for the exploration of personalized medicine and elaborate new drugs.

genomics↗