Biology subjects

Antonescu, C. N.

Publications and source records attributed to Antonescu, C. N..

18 recordsLinked to original sources

bioRxiv2026

Mitophagy is required for cell survival upon ultrasound and microbubble therapeutic exposure

Ultrasound-stimulated microbubble (USMB) sonoporation treatment is an emerging strategy for cancer therapy that enhances drug delivery by perforating the plasma membrane. This membrane permeabilization also causes cellular metabolic stress as cells leak metabolites such as ATP and amino acids. Collectively, cells may die in response to excess membrane damage and stress. However, many cells survive the USMB insult by engaging membrane repair and adaptation pathways such as stimulation of AMPK. In this study, we investigated the impact of USMB treatment on mitochondria and their role in post-exposure cellular survival. We found that USMB causes mitochondrial fragmentation and decreases mitochondrial membrane potential. Subsequently, autophagy and Parkin-dependent mitophagy are triggered post-USMB treatment in an AMPK-dependent mechanism. Importantly, Parkin silencing and PINK1 inhibition augment cell death when combined with USMB exposure, suggesting that mitophagy is an important cell survival process in response to USMB. We propose that combining USMB with inhibitors of mitophagy and metabolic stress adaptation is a strong candidate for therapeutic development to treat solid tumours.

cell biology↗

bioRxiv2026

Spatially localized ligand binding to receptors affects magnitude and timing of signaling response

Cell surface receptors are activated by ligand binding and transmit signals into the cell. Epidermal growth factor (EGF) receptor (EGFR) signaling regulates cell growth, differentiation, and survival, and its dysregulation is linked to cancer. Recent experiments show that ligand binding to EGFR is enhanced for receptors in tetraspanin nanodomains on the cell surface. We use kinetic modeling of receptor confinement, ligand binding, and internalization to compare confinement and signaling behavior for EGFR with spatially localized ligand binding to a hypothetical receptor that has uniform ligand binding anywhere on the cell surface. We find that introducing a membrane domain that confines and enhances ligand binding to receptors leads to more consistent confinement across ligand levels, raises necessary ligand levels for steady-state signaling, and flattens and extends the signaling response to sudden ligand concentration increases. This confining domain that enhances ligand binding provides the cell with a distinct regulatory mechanism to tune its signaling response. We also find that the concentration of receptors in signaling states and the fraction of receptors in signaling states respond to ligand at different ligand concentrations, with substantial increase of the concentration of receptors in signaling states occurring at a much lower ligand concentration than a substantial increase of the fraction of surface receptors in signaling states. This quantitative modeling of spatially restricted receptor activation applies to other receptors with similar characteristics and builds towards physical principles of receptor signaling.

biophysics↗

bioRxiv2025

AMPK Repositions Early Endosomes via Gapex-5 to Promote Delivery of Iron to Mitochondria

The regulation of the spatial organization of organelles within cells is critical for coordinating signaling, membrane traffic, and metabolite exchange. Metabolic cues regulate the position and function of lysosomes, yet whether and how metabolic signals may similarly regulate other organelles such as early endosomes (EEs) remains unclear. We find that AMP-activated protein kinase (AMPK), a key regulator of metabolic homeostasis activated in response to nutrient scarcity, triggers movement of EEs to the perinuclear region of cells, leading to enhanced proximity of endosomes to mitochondria and increased delivery of iron to mitochondria. The movement of EEs and increased mitochondrial iron content elicited by AMPK activation requires Gapex-5, a GEF for the early endosome Rab5 previously shown to be an AMPK substrate. These findings reveal a mechanism by which AMPK reprograms endosome positioning to facilitate inter-organelle communication and iron delivery to mitochondria to support metabolic adaptation under conditions of nutrient scarcity.

cell biology↗

bioRxiv2025

Differences in intrinsic cellular O-GlcNAcylation impact response to metabolic stress

The cellular response to metabolic stress is complex and involves various signals and pathways leading to cellular adaptation. AMP-activated Protein Kinase (AMPK) senses nutrient insufficiency reflected in the ratio of ATP to AMP/ADP and/or glucose availability. In addition, the dynamic modification of proteins with O-linked {beta}-N-Acetylglucosamine (O-GlcNAc) by O-GlcNAc transferase (OGT) is also nutrient sensitive, given that the substrate for this modification is the product of the hexosamine biosynthetic pathway. AMPK can also regulate the hexosamine biosynthetic pathway, and O-GlcNAc may reciprocally regulate AMPK activity under some contexts. However, it remains unclear which parameters establish the extent of reciprocal regulation of AMPK and protein O-GlcNAc modification and whether these signals can be orthogonal under some circumstances. Here, we used ARPE-19 and MDA-MB-231 cells to examine how nutrient limitation or pharmacological activation combined with inhibition of AMPK and O-GlcNAcylation impacts the activation of the reciprocal pathway. We found that the intrinsic level of global protein O-GlcNAc modification may impact how AMPK and metabolic stress regulate global O-GlcNAcylation, and that both signaling systems could be orthogonal in some contexts. These results contribute to understanding the complexity of cellular signaling in response to nutrient availability.

cell biology↗

bioRxiv2025

NCK adaptor proteins regulate clathrin-coated pit dynamics and EGF-stimulated PI3K-Akt signaling

The epidermal growth factor (EGF) receptor (EGFR) promotes cell growth, proliferation, and survival. EGF binding to EGFR activates receptor kinase activity, leading to membrane recruitment of class IA phosphoinositide-3-kinase (PI3K), thus activating Akt signaling. Clathrin-coated pits (CCPs) are plasma membrane endocytic structures that are enriched in signaling intermediates and regulate in EGF-stimulated Akt activation. The mechanisms and impact of recruitment of certain signaling molecules within the PI3K-Akt pathway to CCPs remains poorly understood. Using total internal reflection fluorescence microscopy (TIRFM), we observed that the adaptor proteins Nck1 and Nck2 are enriched within CCPs and regulate early CCP formation and maturation. Nck1 and Nck2 each support EGF-stimulated Akt phosphorylation. Notably, EGF stimulation triggers Nck-dependent enrichment of PI3K within CCPs, and perturbation of Nck adaptors suppressed cell proliferation and survival. This study identifies novel functions for Nck adaptor proteins in EGFR-mediated recruitment of PI3K-Akt signals within CCPs, Akt activation, and cell physiology.

cell biology↗

bioRxiv2024

Cargo-selective regulation of clathrin-mediated endocytosis by AMP-activated protein kinase

The cell surface abundance of many proteins is controlled by clathrin-mediated endocytosis (CME). CME is driven by the assembly of clathrin and other proteins on the inner leaflet of the plasma membrane into clathrin-coated pits (CCPs). Regulation of CCP dynamics allows for control of the function of specific cell surface proteins, impacting a range of cellular outcomes. AMP-activated protein kinase (AMPK) becomes activated upon metabolic insufficiency and facilitates cellular adaptation to nutrient stress. Here, we examined how AMPK regulates CME and the cell surface membrane traffic of {beta}1-integrin. We find that AMPK controls CCP dynamics and regulates the abundance of the endocytic adaptor protein Dab2 within CCPs in a manner that requires the GTPase Arf6, thus selectively promoting the CCP recruitment and internalization of {beta}1-integrin. This reveals a novel signaling pathway for cargo-selective metabolic regulation of CME by AMPK, impacting the function of cell surface proteins such as integrins.

cell biology↗

bioRxiv2024

Control of GSK3beta nuclear localization by amino acid signaling requires GATOR1 but is mTORC1 independent

The availability of certain amino acids regulates cell survival, proliferation, growth, differentiation, and other cellular functions. Sensing of amino acids that converges on the GATOR1 and GATOR2 complexes supports activation of mTORC1 during amino acid replete conditions. Whether amino acid-derived cues regulate additional pathways remains poorly understood. We uncover that amino acid sensing involving GATOR1 and GATOR2 regulates the cellular localization of glycogen synthase kinase 3{beta} (GSK3{beta}). GATOR1 is required to recruit a subset of GSK3{beta} to the lysosome selectively in the presence of amino acids. In addition, while under nutrient replete conditions GSK3{beta} is largely cytosolic, amino acid starvation drives a portion of GSK3{beta} into the nucleus. Acute replenishment of specific amino acids in starved cells triggered nuclear exit of GSK3{beta}. This amino acid-stimulated GSK{beta} nuclear exit required GATOR1 and GATOR2 but was independent of mTORC1 and its activating RagA/B GTPases. This suggests that GATOR1 has a function that diverges from control of mTORC1 to regulate the nucleocytoplasmic shuttling of GSK3{beta}. Furthermore, experimental restriction of GSK3{beta} to the cytoplasm decreased cell survival in amino acid deficient conditions. This suggests that control of GSK3{beta} nuclear localization by GATOR-dependent signals represents a cellular adaptation to metabolic stress that supports cell survival.

cell biology↗

bioRxiv2023

Loss of Tumour Suppressor TMEM127 Drives RET-mediated Transformation Through Disrupted Membrane Dynamics

Internalization from the cell membrane and endosomal trafficking of receptor tyrosine kinases (RTK) are important regulators of signaling in normal cells that can frequently be disrupted in cancer. The adrenal tumour pheochromocytoma (PCC) can be caused by activating mutations of the RET receptor tyrosine kinase, or inactivation of TMEM127, a transmembrane tumour suppressor implicated in trafficking of endosomal cargos. However, the role of aberrant receptor trafficking in PCC is not well understood. Here, we show that loss of TMEM127 causes wildtype RET protein accumulation on the cell surface, where increased receptor density facilitates constitutive ligand-independent activity and downstream signaling, driving cell proliferation. Loss of TMEM127 altered normal cell membrane organization and recruitment and stabilization of membrane protein complexes, impaired assembly, and maturation of clathrin coated pits, and reduced internalization and degradation of cell surface RET. In addition to RTKs, TMEM127 depletion also promoted surface accumulation of several other transmembrane proteins, suggesting it may cause global defects in surface protein activity and function. Together, our data identify TMEM127 as an important determinant of membrane organization including membrane protein diffusability, and protein complex assembly and provide a novel paradigm for oncogenesis in PCC where altered membrane dynamics promotes cell surface accumulation and constitutive activity of growth factor receptors to drive aberrant signaling and promote transformation.

cancer biology↗

bioRxiv2023

Quantitative modeling of EGF receptor ligand discrimination via internalization proofreading

The epidermal growth factor receptor (EGFR) is a central regulator of cell physiology that is stimulated by multiple distinct ligands. Although ligands bind to EGFR while the receptor is exposed on the plasma membrane, EGFR incorporation into endosomes following receptor internalization is an important aspect of EGFR signaling, with EGFR internalization behavior dependent upon the type of ligand bound. We develop quantitative modeling, both kinetic and with spatial details, for EGFR recruitment to and internalization from clathrin domains and competition with ligand unbinding from EGFR. We find that a combination of spatial and kinetic proofreading leads to enhanced EGFR internalization ratios in comparison to unbinding differences between ligand types. Various stages of the EGFR internalization process, including recruitment to and internalization from clathrin domains, modulate the internalization differences between receptors bound to different ligands. Our results indicate that following ligand binding, EGFR may encounter multiple clathrin domains before successful recruitment and internalization. The quantitative modeling we have developed describes competition between EGFR internalization and ligand unbinding and the resulting proofreading.

biophysics↗

bioRxiv2023

The LCLAT1/LYCAT acyltransferase supports EGF-mediated phosphatidylinositol-3,4,5-trisphosphate and Akt signalling

Receptor tyrosine kinases such as epidermal growth factor receptor (EGFR) stimulate phosphoinositide 3-kinases (PI3Ks) to convert phosphatidylinositol-4,5-bisphosophate [PtdIns(4,5)P2] into phosphatidylinositol-3,4,5-trisphosphate [PtdIns(3,4,5)P3]. PtdIns(3,4,5)P3 then remodels actin and gene expression, and boosts cell survival and proliferation. PtdIns(3,4,5)P3 partly achieves these functions by triggering activation of the kinase Akt, which phosphorylates targets like Tsc2 and GSK3{beta}. Consequently, unchecked upregulation of PtdIns(3,4,5)P3-Akt signalling promotes tumour progression. Interestingly, 50-70% of PtdIns and PtdInsPs have stearate and arachidonate at sn-1 and sn-2 positions of glycerol, respectively, forming a species known as 38:4-PtdIns/PtdInsPs. LCLAT1 and MBOAT7 acyltransferases partly enrich PtdIns in this acyl format. We previously showed that disruption of LCLAT1 lowered PtdIns(4,5)P2 levels and perturbed endocytosis and endocytic trafficking. However, the role of LCLAT1 in receptor tyrosine kinase and PtdIns(3,4,5)P3 signaling was not explored. Here, we show that LCLAT1 silencing in MDA-MB-231 and ARPE-19 cells abated the levels of PtdIns(3,4,5)P3 in response to EGF signalling. Importantly, LCLAT1-silenced cells were also impaired for EGF-driven and insulin-driven Akt activation and downstream signalling. Thus, our work provides first evidence that the LCLAT1 acyltransferase is required for receptor tyrosine kinase signalling.

cell biology↗

bioRxiv2022

O-GlcNAc transferase modulates formation of clathrin-coated pits

Clathrin-mediated endocytosis (CME) controls the internalization and function of a wide range of cell surface proteins. CME occurs by the assembly of clathrin and many other proteins on the inner leaflet of the plasma membrane into clathrin-coated pits (CCPs). These structures recruit specific membrane protein cargo destined for internalization and trigger the generation of membrane curvature that precedes eventual scission of CCPs from the plasma membrane to yield intracellular vesicles. The diversity of cell surface protein cargo thus controlled by CME indicates that CCP formation is regulated to allow cellular adaptation under different contexts. Of interest is how cues derived from cellular metabolism may regulate CME, given the reciprocal role of CME in controlling cellular metabolism. The modification of proteins with O-linked {beta}-N-acetylglucosamine (O-GlcNAc) is sensitive to nutrient availability and may allow cellular adaptation to different metabolic conditions. We examined how the modification of proteins with O-GlcNAc may control CCP formation and thus CME. We used perturbation of key enzymes responsible for protein O-GlcNAc modification, as well as specific mutants of the endocytic regulator AAK1 predicted to be impaired for O-GlcNAc modification. We identify that CCP initiation and the assembly of clathrin and other proteins within CCPs is controlled by O-GlcNAc protein modification. This reveals a new dimension of regulation of CME and highlights the important reciprocal regulation of cellular metabolism and endocytosis.

cell biology↗

bioRxiv2022

Improved imaging and preservation of lysosome dynamics using silver nanoparticle-enhanced fluorescence

The dynamics of living cells can be studied by live-cell fluorescence microscopy. However, this requires the use of excessive light energy to obtain good signal-to-noise ratio, which can then photobleach fluorochromes, and more worrisomely, lead to photo-toxicity. Upon light excitation, noble metal nanoparticles such as silver nanoparticles (AgNP) generate plasmons, which can then amplify excitation in direct proximity of the nanoparticles surface and couple to the oscillating dipole of nearby radiating fluorophores, modifying their rate of emission and thus, enhancing their fluorescence. Here, we show that AgNP fed to cells to accumulate within lysosomes enhanced the fluorescence of lysosome-targeted Alexa488-conjugated dextran, BODIPY-cholesterol, and DQ-BSA. Moreover, AgNP increased the fluorescence of GFP fused to the cytosolic tail of LAMP1, showing that metal enhanced fluorescence can occur across the lysosomal membrane. The inclusion of AgNPs in lysosomes did not disturb lysosomal properties such as lysosomal pH, degradative capacity, autophagy and autophagic flux, and membrane integrity, though AgNP seemed to increase basal lysosome tubulation. Importantly, by using AgNP, we could track lysosome motility with reduced laser power without damaging and altering lysosome dynamics. Overall, AgNP-enhanced fluorescence may be a useful tool to study the dynamics of the endo-lysosomal pathway while minimizing photo-toxicity. eTOCSilver nanoparticles enhance fluorescence via surface plasmons. Here, we show that loading lysosomes with silver nanoparticles enhances the fluorescence of fluorochrome- and GFP-based molecular probes for lysosomes. This affords reduced excitation and exposure, diminishing photobleaching and phototoxicity, and preserving lysosome dynamics.

cell biology↗

bioRxiv2022

HER2 expression defines unique requirements for flotillin and c-Src for EGFR signaling

The epidermal growth factor receptor (EGFR) controls many cellular functions. Upon binding its ligand, the receptor undergoes dimerization, phosphorylation, and activation of signals including the phosphatidylinositol-3-kinase (PI3K)-Akt pathway. While some studies indicated that EGFR signaling may be controlled by signal enrichment within membrane raft nanodomains, others have found a limited effect of membrane raft disruption on EGFR signaling, suggesting that specific factor(s) may define context-specific control of EGFR signaling by membrane rafts. Ligand-bound EGFR can homodimerize, or instead undergo heterodimerization with the related receptor HER2 when the latter is expressed. We examined how EGFR signaling in the presence of HER2 distinctly requires membrane raft nanodomains. Induction of HER2 expression altered EGFR signaling duration consistent with EGFR/HER2 heterodimer formation. EGFR and c-Src localized within plasma membrane structures demarked by flotillin, a membrane raft protein, selectively in HER2-expressing cells. Consistently, HER2-expressing cells, but not cells lacking HER2, were dependent on flotillin and c-Src for EGFR signaling leading to Akt activation and cell proliferation. Hence, HER2 expression establishes the requirement of EGFR signaling for flotillin membrane rafts and c-Src, leading to Akt activation. Summary statementThe role of membrane rafts in EGFR signaling may be context-specific. We find that the related receptor HER2 defines unique signaling requirements for EGFR for membrane rafts, flotillin, and c-Src.

cell biology↗

bioRxiv2022

AMP-activated protein kinase (AMPK) is required for recovery from metabolic stress induced by ultrasound microbubble treatment

Ultrasound and microbubbles (USMB) is a promising strategy for cancer therapy. USMB can induce a variety of effects on cells including transient formation of plasma membrane pores (sonoporation) and enhanced endocytosis, which enhance drug delivery, and can also lead to enhanced cell death. However, the outcomes of USMB on cell physiology are heterogeneous, in that USMB elicits cell death in a proportion of cells while exerting minimal effects on others. This suggests that mechanisms of adaptation following USMB allow some cells to survive and/or proliferate. The molecular mechanisms of adaptation to USMB-induced stress remain poorly understood, thus potentially hindering broad therapeutic applications of USMB. Herein, we used several triple negative breast cancer cells to study the effect of USMB-induced metabolite stress and the role of AMPK as a response to this stress. We found that USMB alters steady-state levels of amino acids, glycolytic intermediates, and citric acid cycle intermediates. USMB treatment acutely reduces ATP levels and stimulates AMP-activated protein kinase (AMPK) phosphorylation and activation. Further, AMPK is required to restore ATP levels in cells that survived the initial insult and support cell proliferation post-USMB treatment. These results suggest that AMPK and metabolic perturbations are likely determinants of the anti-neoplastic efficacy of USMB treatment.

cell biology↗

bioRxiv2022

Confinement of unliganded EGFR by tetraspanin nanodomains gates EGFR ligand binding and signaling

The epidermal growth factor receptor (EGFR) is a central regulator of cell physiology. EGFR is activated by ligand binding, triggering receptor dimerization, activation of kinase activity, and intracellular signaling. EGFR is transiently confined within various plasma membrane nanodomains, yet how this may contribute to regulation of EGFR ligand binding is poorly understood. To resolve how EGFR nanoscale compartmentalization gates ligand binding, we developed single-particle tracking methods to track the mobility of ligand-bound and total EGFR, in combination with modeling of EGFR ligand binding. In comparison to unliganded EGFR, ligand-bound EGFR was more confined and distinctly regulated by clathrin and tetraspanin nanodomains. Ligand binding to unliganded EGFR occurred preferentially in tetraspanin nanodomains, and disruption of tetraspanin nanodomains impaired EGFR ligand binding and altered the conformation of the receptors ectodomain. We thus reveal a novel mechanism by which EGFR confinement within tetraspanin nanodomains regulates receptor signaling at the level of ligand binding.

cell biology↗

bioRxiv2021

Multiscale interactome analysis coupled with off-target drug predictions reveals drug repurposing candidates for human coronavirus disease

The COVID-19 pandemic has led to an urgent need for the identification of new antiviral drug therapies that can be rapidly deployed to treat patients with this disease. COVID-19 is caused by infection with the human coronavirus SARS-CoV-2. We developed a computational approach to identify new antiviral drug targets and repurpose clinically-relevant drug compounds for the treatment of COVID-19. Our approach is based on graph convolutional networks (GCN) and involves multiscale host-virus interactome analysis coupled to off-target drug predictions. Cellbased experimental assessment reveals several clinically-relevant repurposing drug candidates predicted by the in silico analyses to have antiviral activity against human coronavirus infection. In particular, we identify the MET inhibitor capmatinib as having potent and broad antiviral activity against several coronaviruses in a MET-independent manner, as well as novel roles for host cell proteins such as IRAK1/4 in supporting human coronavirus infection, which can inform further drug discovery studies.

cell biology↗

bioRxiv2020

Fatty Acid Synthase inhibition prevents palmitoylation of SARS-CoV2 SpikeProtein and improves survival of mice infected with murine hepatitis virus.

The Spike protein of SARS-CoV2 and other coronaviruses mediate host cell entry and are S-acylated on multiple phylogenetically conserved cysteine residues. Multiple protein acyltransferase enzymes of the ZDHHC family have been reported to modify Spike proteins post-translationally. Using resin-assisted capture mass spectrometry, we demonstrate that the Spike protein is S-acylated in SARS-CoV2 infected human and monkey cells. We further show that increased abundance of the human acyltransferase ZDHHC5 results in increased S-acylation of the SARS-CoV2 Spike protein, whereas ZDHHC5 knockout cells had a 40% reduction in the incorporation of an alkynyl-palmitate using click chemistry detection. We also find that the S-acylation of the Spike protein is not limited to palmitate, as clickable versions of myristate and stearate were also found on the immunocaptured protein. Yet, ZDHHC5 was highly selective for palmitate, suggesting that other ZDHHC enzymes mediated the incorporation of other fatty acyl chains. Thus, since multiple ZDHHC isoforms may modify the Spike protein, we examined the ability of the fatty acid synthase inhibitor TVB-3166 to prevent the S-acylation of the Spike proteins of SARS-CoV-2 and human CoV-229E. Treating cells with TVB-3166 inhibited S-acylation of ectopically expressed SARS-CoV2 Spike and attenuated the ability of SARS-CoV2 and human CoV-229E to spread in vitro. Additionally, treatment of mice with a comparatively low dose of TVB-3166 promoted survival from an otherwise fatal murine coronavirus infection. Our findings further substantiate the necessity of CoV Spike protein S-acylation and the potential use of fatty acid synthase inhibitors.

cell biology↗

bioRxiv2018

mTORC1 controls glycogen synthase kinase 3β nuclear localization and function

Glycogen synthase kinase 3{beta} (GSK3{beta}) phosphorylates and regulates a wide range of substrates involved in diverse cellular functions. Some GSK3{beta} substrates, such as c-myc and snail, are nuclear-resident transcription factors, suggesting possible control of GSK3{beta} function by regulation of its nuclear localization. Inhibition of mechanistic target of rapamycin (mTORC1) led to partial redistribution of GSK3{beta} from the cytosol to the nucleus, and GSK3{beta}-dependent reduction of the expression of c-myc and snail. mTORC1 is controlled by metabolic cues, such as by AMP-activated protein kinase (AMPK) or amino acid abundance. Indeed AMPK activation or amino acid deprivation promoted GSK3{beta} nuclear localization in an mTORC1-dependent manner. GSK3{beta} was detected in several distinct endomembrane compartments, including lysosomes. Consistently, disruption of late endosomes/lysosomes through perturbation of Rab7 resulted in loss of GSK3{beta} from lysosomes, and enhanced GSK3{beta} nuclear localization as well as GSK3{beta}-dependent reduction of c-myc levels. This indicates that GSK3{beta} nuclear localization and function is suppressed by mTORC1, and suggests a new link between metabolic conditions sensed by mTORC1 and GSK3{beta}-dependent regulation of transcriptional networks controlling biomass production.\n\nSummary statement (15-30 words)GSK3{beta} nuclear localization and function is negatively regulated by the metabolic and mitogenic sensor mTORC1. mTORC1 control of GSK3{beta} localization requires Rab7 and lysosomal membrane traffic.

cell biology↗