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Horwath O, Moberg M, Hodson N, Edman S, Johansson M, Andersson E, van Hall G, Rooyackers O, Philp A, Apró W. Anabolic Sensitivity in Healthy, Lean, Older Men Is Associated With Higher Expression of Amino Acid Sensors and mTORC1 Activators Compared to Young. J Cachexia Sarcopenia Muscle 2025; 16:e13613. [PMID: 39558870 DOI: 10.1002/jcsm.13613] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 06/20/2024] [Revised: 09/01/2024] [Accepted: 09/11/2024] [Indexed: 11/20/2024] Open
Abstract
BACKGROUND Sarcopenia is thought to be underlined by age-associated anabolic resistance and dysregulation of intracellular signalling pathways. However, it is unclear whether these phenomena are driven by ageing per se or other confounding factors. METHODS Lean and healthy young (n = 10, 22 ± 3 years, BMI; 23.4 ± 0.8 kg/m2) and old men (n = 10, 70 ± 3 years, BMI; 22.7 ± 1.3 kg/m2) performed unilateral resistance exercise followed by intake of essential amino acids (EAA). Muscle biopsies were collected from the rested and the exercised leg before, immediately after and 60 and 180 min after EAA intake. Muscle samples were analysed for amino acid concentrations, muscle protein synthesis (MPS) and associated anabolic signalling. RESULTS Following exercise, peak plasma levels of EAA and leucine were similar between groups, but the area under the curve was ~11% and ~28% lower in Young (p < 0.01). Absolute levels of muscle EAA and leucine peaked 60 min after exercise, with ~15 and ~21% higher concentrations in the exercising leg (p < 0.01) but with no difference between groups. MPS increased in both the resting (~0.035%·h-1 to 0.056%·h-1, p < 0.05) and exercising leg (~0.035%·h-1 to 0.083%·h-1, p < 0.05) with no difference between groups. Phosphorylation of S6K1Thr389 increased to a similar extent in the exercising leg in both groups but was 2.8-fold higher in the resting leg of Old at the 60 min timepoint (p < 0.001). Phosphorylation of 4E-BP1Ser65 increased following EAA intake and exercise, but differences between legs were statistically different only at 180 min (p < 0.001). However, phosphorylation of this site was on average 78% greater across all timepoints in Old (p < 0.01). Phosphorylation of eEF2Thr56 was reduced (~66% and 39%) in the exercising leg at both timepoints after EAA intake and exercise, with no group differences (p < 0.05). However, phosphorylation at this site was reduced by ~27% also in the resting leg at 60 min, an effect that was only seen in Old (p < 0.01). Total levels of Rheb (~45%), LAT1 (~31%) and Rag B (~31%) were higher in Old (p < 0.001). CONCLUSION Lean and healthy old men do not manifest AR as evidenced by potent increases in MPS and mTORC1 signalling following EAA intake and exercise. Maintained anabolic sensitivity with age appears to be a function of a compensatory increase in basal levels of proteins involved in anabolic signalling. Therefore, our results suggest that age per se does not appear to cause AR in human skeletal muscle.
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Affiliation(s)
- Oscar Horwath
- Department of Physiology, Nutrition and Biomechanics, The Swedish School of Sport and Health Sciences, Stockholm, Sweden
| | - Marcus Moberg
- Department of Physiology, Nutrition and Biomechanics, The Swedish School of Sport and Health Sciences, Stockholm, Sweden
- Department of Physiology and Pharmacology, Karolinska Institute, Stockholm, Sweden
| | - Nathan Hodson
- Department of Exercise Sciences, Faculty of Kinesiology and Physical Education, University of Toronto, Toronto, Ontario, Canada
- Department of Sport and Exercise Sciences, Institute of Sport, Manchester Metropolitan University, Manchester, UK
| | - Sebastian Edman
- Department of Physiology, Nutrition and Biomechanics, The Swedish School of Sport and Health Sciences, Stockholm, Sweden
- Department of Women's and Children's Health, Karolinska Institute, Stockholm, Sweden
| | - Mats Johansson
- Division of Clinical Chemistry, Department of Laboratory Medicine, Karolinska Institute, Stockholm, Sweden
| | - Eva Andersson
- Department of Physiology, Nutrition and Biomechanics, The Swedish School of Sport and Health Sciences, Stockholm, Sweden
- Department of Molecular Medicine and Surgery, Karolinska Institute, Stockholm, Sweden
| | - Gerrit van Hall
- Department of Biomedical Sciences, Faculty of Health and Medical Sciences, University of Copenhagen, Copenhagen, Denmark
- Clinical Metabolomics Core Facility, Department of Clinical Biochemistry, Rigshospitalet, University of Copenhagen, Copenhagen, Denmark
| | - Olav Rooyackers
- Department of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden
| | - Andrew Philp
- Centre for Healthy Ageing, Centenary Institute, Sydney, New South Wales, Australia
- School of Sport, Exercise and Rehabilitation Sciences, University of Technology Sydney, Sydney, New South Wales, Australia
| | - William Apró
- Department of Physiology, Nutrition and Biomechanics, The Swedish School of Sport and Health Sciences, Stockholm, Sweden
- Department of Clinical Science, Intervention and Technology, Karolinska Institute, Stockholm, Sweden
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Gest AM, Sahan AZ, Zhong Y, Lin W, Mehta S, Zhang J. Molecular Spies in Action: Genetically Encoded Fluorescent Biosensors Light up Cellular Signals. Chem Rev 2024; 124:12573-12660. [PMID: 39535501 PMCID: PMC11613326 DOI: 10.1021/acs.chemrev.4c00293] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 04/17/2024] [Revised: 09/07/2024] [Accepted: 09/20/2024] [Indexed: 11/16/2024]
Abstract
Cellular function is controlled through intricate networks of signals, which lead to the myriad pathways governing cell fate. Fluorescent biosensors have enabled the study of these signaling pathways in living systems across temporal and spatial scales. Over the years there has been an explosion in the number of fluorescent biosensors, as they have become available for numerous targets, utilized across spectral space, and suited for various imaging techniques. To guide users through this extensive biosensor landscape, we discuss critical aspects of fluorescent proteins for consideration in biosensor development, smart tagging strategies, and the historical and recent biosensors of various types, grouped by target, and with a focus on the design and recent applications of these sensors in living systems.
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Affiliation(s)
- Anneliese
M. M. Gest
- Department
of Pharmacology, University of California,
San Diego, La Jolla, California 92093, United States
| | - Ayse Z. Sahan
- Department
of Pharmacology, University of California,
San Diego, La Jolla, California 92093, United States
- Biomedical
Sciences Graduate Program, University of
California, San Diego, La Jolla, California 92093, United States
| | - Yanghao Zhong
- Department
of Pharmacology, University of California,
San Diego, La Jolla, California 92093, United States
| | - Wei Lin
- Department
of Pharmacology, University of California,
San Diego, La Jolla, California 92093, United States
| | - Sohum Mehta
- Department
of Pharmacology, University of California,
San Diego, La Jolla, California 92093, United States
| | - Jin Zhang
- Department
of Pharmacology, University of California,
San Diego, La Jolla, California 92093, United States
- Shu
Chien-Gene Lay Department of Bioengineering, University of California, San Diego, La Jolla, California 92093, United States
- Department
of Chemistry and Biochemistry, University
of California, San Diego, La Jolla, California 92093, United States
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Fernandes SA, Angelidaki DD, Nüchel J, Pan J, Gollwitzer P, Elkis Y, Artoni F, Wilhelm S, Kovacevic-Sarmiento M, Demetriades C. Spatial and functional separation of mTORC1 signalling in response to different amino acid sources. Nat Cell Biol 2024; 26:1918-1933. [PMID: 39385049 PMCID: PMC11567901 DOI: 10.1038/s41556-024-01523-7] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 02/01/2024] [Accepted: 09/09/2024] [Indexed: 10/11/2024]
Abstract
Amino acid (AA) availability is a robust determinant of cell growth through controlling mechanistic/mammalian target of rapamycin complex 1 (mTORC1) activity. According to the predominant model in the field, AA sufficiency drives the recruitment and activation of mTORC1 on the lysosomal surface by the heterodimeric Rag GTPases, from where it coordinates the majority of cellular processes. Importantly, however, the teleonomy of the proposed lysosomal regulation of mTORC1 and where mTORC1 acts on its effector proteins remain enigmatic. Here, by using multiple pharmacological and genetic means to perturb the lysosomal AA-sensing and protein recycling machineries, we describe the spatial separation of mTORC1 regulation and downstream functions in mammalian cells, with lysosomal and non-lysosomal mTORC1 phosphorylating distinct substrates in response to different AA sources. Moreover, we reveal that a fraction of mTOR localizes at lysosomes owing to basal lysosomal proteolysis that locally supplies new AAs, even in cells grown in the presence of extracellular nutrients, whereas cytoplasmic mTORC1 is regulated by exogenous AAs. Overall, our study substantially expands our knowledge about the topology of mTORC1 regulation by AAs and hints at the existence of distinct, Rag- and lysosome-independent mechanisms that control its activity at other subcellular locations. Given the importance of mTORC1 signalling and AA sensing for human ageing and disease, our findings will probably pave the way towards the identification of function-specific mTORC1 regulators and thus highlight more effective targets for drug discovery against conditions with dysregulated mTORC1 activity in the future.
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Affiliation(s)
- Stephanie A Fernandes
- Max Planck Institute for Biology of Ageing, Cologne, Germany
- Cologne Graduate School of Ageing Research, Cologne, Germany
| | | | - Julian Nüchel
- Max Planck Institute for Biology of Ageing, Cologne, Germany
- Center for Biochemistry, Medical Faculty, University of Cologne, Cologne, Germany
| | - Jiyoung Pan
- Max Planck Institute for Biology of Ageing, Cologne, Germany
- Cologne Graduate School of Ageing Research, Cologne, Germany
| | | | - Yoav Elkis
- Max Planck Institute for Biology of Ageing, Cologne, Germany
| | - Filippo Artoni
- Max Planck Institute for Biology of Ageing, Cologne, Germany
- Cologne Graduate School of Ageing Research, Cologne, Germany
| | - Sabine Wilhelm
- Max Planck Institute for Biology of Ageing, Cologne, Germany
| | | | - Constantinos Demetriades
- Max Planck Institute for Biology of Ageing, Cologne, Germany.
- Cologne Graduate School of Ageing Research, Cologne, Germany.
- Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases, University of Cologne, Cologne, Germany.
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Romanyuk N, Sintakova K, Arzhanov I, Horak M, Gandhi C, Jhanwar-Uniyal M, Jendelova P. mTOR pathway inhibition alters proliferation as well as differentiation of neural stem cells. Front Cell Neurosci 2024; 18:1298182. [PMID: 38812794 PMCID: PMC11133533 DOI: 10.3389/fncel.2024.1298182] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/21/2023] [Accepted: 04/23/2024] [Indexed: 05/31/2024] Open
Abstract
Introduction Neural stem cells (NSCs) are essential for both embryonic development and adult neurogenesis, and their dysregulation causes a number of neurodevelopmental disorders, such as epilepsy and autism spectrum disorders. NSC proliferation and differentiation in the developing brain is a complex process controlled by various intrinsic and extrinsic stimuli. The mammalian target of rapamycin (mTOR) regulates proliferation and differentiation, among other cellular functions, and disruption in the mTOR pathway can lead to severe nervous system development deficits. In this study, we investigated the effect of inhibition of the mTOR pathway by rapamycin (Rapa) on NSC proliferation and differentiation. Methods The NSC cultures were treated with Rapa for 1, 2, 6, 24, and 48 h. The effect on cellular functions was assessed by immunofluorescence staining, western blotting, and proliferation/metabolic assays. Results mTOR inhibition suppressed NSC proliferation/metabolic activity as well as S-Phase entry by as early as 1 h of Rapa treatment and this effect persisted up to 48 h of Rapa treatment. In a separate experiment, NSCs were differentiated for 2 weeks after treatment with Rapa for 24 or 48 h. Regarding the effect on neuronal and glial differentiation (2 weeks post-treatment), this was suppressed in NSCs deficient in mTOR signaling, as evidenced by downregulated expression of NeuN, MAP2, and GFAP. We assume that the prolonged effect of mTOR inhibition is realized due to the effect on cytoskeletal proteins. Discussion Here, we demonstrate for the first time that the mTOR pathway not only regulates NSC proliferation but also plays an important role in NSC differentiation into both neuronal and glial lineages.
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Affiliation(s)
- Nataliya Romanyuk
- Department of Neuroregeneration, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czechia
| | - Kristyna Sintakova
- Department of Neuroregeneration, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czechia
- Department of Neuroscience, Second Faculty of Medicine, Charles University, Prague, Czechia
| | - Ivan Arzhanov
- Department of Neuroregeneration, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czechia
- Department of Neuroscience, Second Faculty of Medicine, Charles University, Prague, Czechia
| | - Martin Horak
- Department of Neurochemistry, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czechia
| | - Chirag Gandhi
- Department of Neurosurgery, New York Medical College, Valhalla, NY, United States
| | - Meena Jhanwar-Uniyal
- Department of Neurosurgery, New York Medical College, Valhalla, NY, United States
| | - Pavla Jendelova
- Department of Neuroregeneration, Institute of Experimental Medicine of the Czech Academy of Sciences, Prague, Czechia
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Wang Y, Engel T, Teng X. Post-translational regulation of the mTORC1 pathway: A switch that regulates metabolism-related gene expression. BIOCHIMICA ET BIOPHYSICA ACTA. GENE REGULATORY MECHANISMS 2024; 1867:195005. [PMID: 38242428 DOI: 10.1016/j.bbagrm.2024.195005] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 06/27/2023] [Revised: 12/15/2023] [Accepted: 01/03/2024] [Indexed: 01/21/2024]
Abstract
The mechanistic target of rapamycin complex 1 (mTORC1) is a kinase complex that plays a crucial role in coordinating cell growth in response to various signals, including amino acids, growth factors, oxygen, and ATP. Activation of mTORC1 promotes cell growth and anabolism, while its suppression leads to catabolism and inhibition of cell growth, enabling cells to withstand nutrient scarcity and stress. Dysregulation of mTORC1 activity is associated with numerous diseases, such as cancer, metabolic disorders, and neurodegenerative conditions. This review focuses on how post-translational modifications, particularly phosphorylation and ubiquitination, modulate mTORC1 signaling pathway and their consequential implications for pathogenesis. Understanding the impact of phosphorylation and ubiquitination on the mTORC1 signaling pathway provides valuable insights into the regulation of cellular growth and potential therapeutic targets for related diseases.
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Affiliation(s)
- Yitao Wang
- College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215123, China; Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, Dublin D02 YN77, Ireland
| | - Tobias Engel
- Department of Physiology and Medical Physics, Royal College of Surgeons in Ireland, University of Medicine and Health Sciences, Dublin D02 YN77, Ireland; FutureNeuro, SFI Research Centre for Chronic and Rare Neurological Diseases, RCSI University of Medicine and Health Sciences, Dublin D02 YN77, Ireland
| | - Xinchen Teng
- College of Pharmaceutical Sciences, Soochow University, Suzhou, Jiangsu 215123, China.
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6
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Jhanwar-Uniyal M, Zeller SL, Spirollari E, Das M, Hanft SJ, Gandhi CD. Discrete Mechanistic Target of Rapamycin Signaling Pathways, Stem Cells, and Therapeutic Targets. Cells 2024; 13:409. [PMID: 38474373 PMCID: PMC10930964 DOI: 10.3390/cells13050409] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/16/2024] [Revised: 02/14/2024] [Accepted: 02/22/2024] [Indexed: 03/14/2024] Open
Abstract
The mechanistic target of rapamycin (mTOR) is a serine/threonine kinase that functions via its discrete binding partners to form two multiprotein complexes, mTOR complex 1 and 2 (mTORC1 and mTORC2). Rapamycin-sensitive mTORC1, which regulates protein synthesis and cell growth, is tightly controlled by PI3K/Akt and is nutrient-/growth factor-sensitive. In the brain, mTORC1 is also sensitive to neurotransmitter signaling. mTORC2, which is modulated by growth factor signaling, is associated with ribosomes and is insensitive to rapamycin. mTOR regulates stem cell and cancer stem cell characteristics. Aberrant Akt/mTOR activation is involved in multistep tumorigenesis in a variety of cancers, thereby suggesting that the inhibition of mTOR may have therapeutic potential. Rapamycin and its analogues, known as rapalogues, suppress mTOR activity through an allosteric mechanism that only suppresses mTORC1, albeit incompletely. ATP-catalytic binding site inhibitors are designed to inhibit both complexes. This review describes the regulation of mTOR and the targeting of its complexes in the treatment of cancers, such as glioblastoma, and their stem cells.
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Affiliation(s)
- Meena Jhanwar-Uniyal
- Department of Neurosurgery, Westchester Medical Center, New York Medical College, Valhalla, NY 10595, USA
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Green MJ, Ge H, Flower SE, Pourzand C, Botchway SW, Wang HC, Kuganathan N, Kociok-Köhn G, Li M, Xu S, James TD, Pascu SI. Fluorescent naphthalimide boronates as theranostics: structural investigations, confocal fluorescence and multiphoton fluorescence lifetime imaging microscopy in living cells. RSC Chem Biol 2023; 4:1082-1095. [PMID: 38033726 PMCID: PMC10685793 DOI: 10.1039/d3cb00112a] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Grants] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/30/2023] [Accepted: 09/17/2023] [Indexed: 12/02/2023] Open
Abstract
New design and synthetic strategies were developed to generate functional phenyl boronic acid (BA)-based fluorescent probes incorporating the 1,8-naphthalimide (NI) tag. This fluorescent core was anchored onto the BA unit through small organic linkers consisting of nitrogen groups which can arrest, and internally stabilise the phenyl-boronate units. The newly synthesised fluorophores were characterised spectroscopically by NMR spectroscopy and mass spectrometry and evaluated for their ability to bind to a naturally occurring polysaccharide, β-d-glucan in DMSO and simultaneously as act as in vitro cell imaging reagents. The uptake of these new NI-boronic acid derivatives was studied living cancer cells (HeLa, PC-3) in the presence, and absence, of β-d-glucan. Time-correlated single-photon counting (TCSPC) of DMSO solutions and two-photon fluorescence-lifetime imaging microscopy (FLIM) techniques allowed an insight into the probes' interaction with their environment. Their cellular uptake and distributions were imaged using laser scanning confocal fluorescence microscopy under single- and two-photon excitation regimes (λmax 910 nm). FLIM facilitated the estimation of the impact of the probe's cellular surroundings using the fluorophore lifetime. The extent to which this was mediated by the β-d-glucan was visualised by 2-photon FLIM in living cells. The fluorescence lifetime observed under a range of temperatures varied appreciably, indicating that changes in the environment can be sensed by these probes. In all cases, the cellular membrane penetration of these new probes was remarkable even under variable temperature conditions and localisation was widely concentrated in the cellular cytoplasm, without specific organelle trapping: we conclude that these new probes show promise for cellular imaging in living cancer cells.
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Affiliation(s)
- Megan J Green
- Department of Chemistry, University of Bath Calverton Down Bath BA2 7AY UK
| | - Haobo Ge
- Department of Chemistry, University of Bath Calverton Down Bath BA2 7AY UK
| | - Stephen E Flower
- Department of Chemistry, University of Bath Calverton Down Bath BA2 7AY UK
| | - Charareh Pourzand
- Department of Life Sciences, University of Bath BA2 7AY UK
- Centre for Therapeutic Innovation, University of Bath BA2 7AY UK
| | - Stanley W Botchway
- STFC Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell Science and Innovation Campus Harwell Oxfordshire OX11 0QX UK
| | - Hui-Chen Wang
- Department of Chemistry, University of Bath Calverton Down Bath BA2 7AY UK
| | | | - Gabriele Kociok-Köhn
- Department of Chemistry, University of Bath Calverton Down Bath BA2 7AY UK
- Materials and Chemical Characterisation Facility (MC2), University of Bath Calverton Down Bath BA2 7AY UK
| | - Meng Li
- Department of Chemistry, University of Bath Calverton Down Bath BA2 7AY UK
- State Key Laboratory of Chemical Resource Engineering, Beijing University of Chemical Technology Beijing 100029 P. R. China
| | - Suying Xu
- Department of Chemistry, University of Bath Calverton Down Bath BA2 7AY UK
- Hebei Key Lab of Power Plant Flue Gas Multi-Pollutants Control, Department of Environmental Science and Engineering, North China Electric Power University Baoding 071003 P. R. China
| | - Tony D James
- Department of Chemistry, University of Bath Calverton Down Bath BA2 7AY UK
| | - Sofia I Pascu
- Department of Chemistry, University of Bath Calverton Down Bath BA2 7AY UK
- Centre for Therapeutic Innovation, University of Bath BA2 7AY UK
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Majeed ST, Majeed R, Malik AA, Andrabi KI. MTORC2 is a physiological hydrophobic motif kinase of S6 Kinase 1. BIOCHIMICA ET BIOPHYSICA ACTA. MOLECULAR CELL RESEARCH 2023; 1870:119449. [PMID: 36858209 DOI: 10.1016/j.bbamcr.2023.119449] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.5] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Subscribe] [Scholar Register] [Received: 12/10/2022] [Revised: 02/16/2023] [Accepted: 02/17/2023] [Indexed: 03/03/2023]
Abstract
Ribosomal protein S6 kinase 1 (S6K1), a major downstream effector molecule of mTORC1, regulates cell growth and proliferation by modulating protein translation and ribosome biogenesis. We have recently identified eIF4E as an intermediate in transducing signals from mTORC1 to S6K1 and further demonstrated that the role of mTORC1 is restricted to inducing eIF4E phosphorylation and interaction with S6K1. This interaction relieves S6K1 auto-inhibition and facilitates its hydrophobic motif (HM) phosphorylation and activation as a consequence. These observations underscore a possible involvement of mTORC1 independent kinase in mediating HM phosphorylation. Here, we report mTORC2 as an in-vivo/physiological HM kinase of S6K1. We show that rapamycin-resistant S6K1 truncation mutant ∆NH∆CT continues to display HM phosphorylation with selective sensitivity toward Torin-1. We also show that HM phosphorylation of wildtype S6K1and ∆NH∆CT depends on the presence of mTORC2 regulatory subunit-rictor. Furthermore, truncation mutagenesis and molecular docking analysis reveal the involvement of a conserved 19 amino acid stretch of S6K1 in mediating interaction with rictor. We finally show that deletion of the 19 amino acid region from wildtype S6K1 results in loss of interaction with rictor, with a resultant loss of HM phosphorylation regardless of the presence of functional TOS motif. Our data demonstrate that mTORC2 acts as a physiological HM kinase that can activate S6K1 after its auto-inhibition is overcome by mTORC1. We, therefore, propose a novel mechanism for S6K1 regulation where mTOR complexes 1 and 2 act in tandem to activate the enzyme.
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Affiliation(s)
- Sheikh Tahir Majeed
- Growth Factor Signaling Laboratory, Department of Biotechnology, University of Kashmir, Srinagar, India; Department of Biotechnology, Central University of Kashmir, Ganderbal, India
| | - Rabiya Majeed
- Growth Factor Signaling Laboratory, Department of Biotechnology, University of Kashmir, Srinagar, India; Department of Biochemistry, University of Kashmir, Srinagar, India
| | - Aijaz A Malik
- Centre of Excellence in Computational Molecular Biology, Faculty of Medicine, Chulalongkorn University, Bangkok, Thailand
| | - Khurshid Iqbal Andrabi
- Growth Factor Signaling Laboratory, Department of Biotechnology, University of Kashmir, Srinagar, India.
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Fluorescence and phosphorescence lifetime imaging reveals a significant cell nuclear viscosity and refractive index changes upon DNA damage. Sci Rep 2023; 13:422. [PMID: 36624137 PMCID: PMC9829731 DOI: 10.1038/s41598-022-26880-x] [Citation(s) in RCA: 13] [Impact Index Per Article: 6.5] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 09/26/2022] [Accepted: 12/20/2022] [Indexed: 01/11/2023] Open
Abstract
Cytoplasmic viscosity is a crucial parameter in determining rates of diffusion-limited reactions. Changes in viscosity are associated with several diseases, whilst nuclear viscosity determines gene integrity, regulation and expression. Yet how drugs including DNA-damaging agents affect viscosity is unknown. We demonstrate the use of a platinum complex, Pt[L]Cl, that localizes efficiently mostly in the nucleus as a probe for nuclear viscosity. The phosphorescence lifetime of Pt[L]Cl is sensitive to viscosity and provides an excellent tool to investigate the impact of DNA damage. We show using Fluorescence Lifetime Imaging (FLIM) that the lifetime of both green and red fluorescent proteins (FP) are also sensitive to changes in cellular viscosity and refractive index. However, Pt[L]Cl proved to be a more sensitive viscosity probe, by virtue of microsecond phosphorescence lifetime versus nanosecond fluorescence lifetime of FP, hence greater sensitivity to bimolecular reactions. DNA damage was inflicted by either a two-photon excitation, one-photon excitation microbeam and X-rays. DNA damage of live cells causes significant increase in the lifetime of either Pt[L]Cl (HeLa cells, 12.5-14.1 µs) or intracellularly expressed mCherry (HEK293 cells, 1.54-1.67 ns), but a decrease in fluorescence lifetime of GFP from 2.65 to 2.29 ns (in V15B cells). These values represent a viscosity change from 8.59 to 20.56 cP as well as significant changes in the refractive index (RI), according to independent calibration. Interestingly DNA damage localized to a submicron region following a laser microbeam induction showed a whole cell viscosity change, with those in the nucleus being greater than the cytoplasm. We also found evidence of a by-stander effect, whereby adjacent un-irradiated cells also showed nuclear viscosity change. Finally, an increase in viscosity following DNA damage was also observed in bacterial cells with an over-expressed mNeonGreen FP, evidenced by the change in its lifetime from 2.8 to 2.4 ns.
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p70 S6 kinase as a therapeutic target in cancers: More than just an mTOR effector. Cancer Lett 2022; 535:215593. [PMID: 35176419 DOI: 10.1016/j.canlet.2022.215593] [Citation(s) in RCA: 17] [Impact Index Per Article: 5.7] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/26/2021] [Revised: 01/25/2022] [Accepted: 02/06/2022] [Indexed: 11/23/2022]
Abstract
p70 S6 kinase (p70S6K) is best-known for its regulatory roles in protein synthesis and cell growth by phosphorylating its primary substrate, ribosomal protein S6, upon mitogen stimulation. The enhanced expression/activation of p70S6K has been correlated with poor prognosis in some cancer types, suggesting that it may serve as a biomarker for disease monitoring. p70S6K is a critical downstream effector of the oncogenic PI3K/Akt/mTOR pathway and its activation is tightly regulated by an ordered cascade of Ser/Thr phosphorylation events. Nonetheless, it should be noted that other upstream mechanisms regulating p70S6K at both the post-translational and post-transcriptional levels also exist. Activated p70S6K could promote various aspects of cancer progression such as epithelial-mesenchymal transition, cancer stemness and drug resistance. Importantly, novel evidence showing that p70S6K may also regulate different cellular components in the tumor microenvironment will be discussed. Therapeutic targeting of p70S6K alone or in combination with traditional chemotherapies or other microenvironmental-based drugs such as immunotherapy may represent promising approaches against cancers with aberrant p70S6K signaling. Currently, the only clinically available p70S6K inhibitors are rapamycin analogs (rapalogs) which target mTOR. However, there are emerging p70S6K-selective drugs which are going through active preclinical or clinical trial phases. Moreover, various screening strategies have been used for the discovery of novel p70S6K inhibitors, hence bringing new insights for p70S6K-targeted therapy.
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Sihag J, Di Marzo V. (Wh)olistic (E)ndocannabinoidome-Microbiome-Axis Modulation through (N)utrition (WHEN) to Curb Obesity and Related Disorders. Lipids Health Dis 2022; 21:9. [PMID: 35027074 PMCID: PMC8759188 DOI: 10.1186/s12944-021-01609-3] [Citation(s) in RCA: 22] [Impact Index Per Article: 7.3] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Grants] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/14/2021] [Accepted: 12/05/2021] [Indexed: 02/06/2023] Open
Abstract
The discovery of the endocannabinoidome (eCBome) is evolving gradually with yet to be elucidated functional lipid mediators and receptors. The diet modulates these bioactive lipids and the gut microbiome, both working in an entwined alliance. Mounting evidence suggests that, in different ways and with a certain specialisation, lipid signalling mediators such as N-acylethanolamines (NAEs), 2-monoacylglycerols (2-MAGs), and N-acyl-amino acids (NAAs), along with endocannabinoids (eCBs), can modulate physiological mechanisms underpinning appetite, food intake, macronutrient metabolism, pain sensation, blood pressure, mood, cognition, and immunity. This knowledge has been primarily utilised in pharmacology and medicine to develop many drugs targeting the fine and specific molecular pathways orchestrating eCB and eCBome activity. Conversely, the contribution of dietary NAEs, 2-MAGs and eCBs to the biological functions of these molecules has been little studied. In this review, we discuss the importance of (Wh) olistic (E)ndocannabinoidome-Microbiome-Axis Modulation through (N) utrition (WHEN), in the management of obesity and related disorders.
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Affiliation(s)
- Jyoti Sihag
- Faculty of Medicine, University of Laval, Quebec, Canada.
- Faculty of Agriculture and Food Sciences, University of Laval, Quebec, Canada.
- Canada Excellence Research Chair on the Microbiome-Endocannabinoidome Axis in Metabolic Health (CERC-MEND), University of Laval, Quebec, Canada.
- University Institute of Cardiology and Pneumology, Quebec, Canada.
- Institute of Nutrition and Functional Foods (INAF) and Centre Nutrition, Santé et Société (NUTRISS), University of Laval, Quebec, Canada.
- Department of Foods and Nutrition, Chaudhary Charan Singh Haryana Agricultural University, Hisar, India.
| | - Vincenzo Di Marzo
- Faculty of Medicine, University of Laval, Quebec, Canada.
- Faculty of Agriculture and Food Sciences, University of Laval, Quebec, Canada.
- Canada Excellence Research Chair on the Microbiome-Endocannabinoidome Axis in Metabolic Health (CERC-MEND), University of Laval, Quebec, Canada.
- University Institute of Cardiology and Pneumology, Quebec, Canada.
- Institute of Nutrition and Functional Foods (INAF) and Centre Nutrition, Santé et Société (NUTRISS), University of Laval, Quebec, Canada.
- Institute of Biomolecular Chemistry of the National Research Council (ICB-CNR), Naples, Italy.
- Endocannabinoid Research Group, Naples, Italy.
- Joint International Research Unit between the Italian National Research Council (CNR) and University of Laval, for Chemical and Biomolecular Research on the Microbiome and its impact on Metabolic Health and Nutrition (UMI-MicroMeNu), Quebec, Canada.
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12
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Arabidopsis thaliana myosin XIK is recruited to the Golgi through interaction with a MyoB receptor. Commun Biol 2021; 4:1182. [PMID: 34645991 PMCID: PMC8514473 DOI: 10.1038/s42003-021-02700-2] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 07/16/2020] [Accepted: 08/31/2021] [Indexed: 12/03/2022] Open
Abstract
Plant cell organelles are highly mobile and their positioning play key roles in plant growth, development and responses to changing environmental conditions. Movement is acto-myosin dependent. Despite controlling the dynamics of several organelles, myosin and myosin receptors identified so far in Arabidopsis thaliana generally do not localise to the organelles whose movement they control, raising the issue of how specificity is determined. Here we show that a MyoB myosin receptor, MRF7, specifically localises to the Golgi membrane and affects its movement. Myosin XI-K was identified as a putative MRF7 interactor through mass spectrometry analysis. Co-expression of MRF7 and XI-K tail triggers the relocation of XI-K to the Golgi, linking a MyoB/myosin complex to a specific organelle in Arabidopsis. FRET-FLIM confirmed the in vivo interaction between MRF7 and XI-K tail on the Golgi and in the cytosol, suggesting that myosin/myosin receptor complexes perhaps cycle on and off organelle membranes. This work supports a traditional mechanism for organelle movement where myosins bind to receptors and adaptors on the organelle membranes, allowing them to actively move on the actin cytoskeleton, rather than passively in the recently proposed cytoplasmic streaming model. Perico et al. use co-expression analysis and a FRET-FLIM approach to show that the Arabidopsis MyoB myosin receptor, MRF7, triggers the relocation of Myosin XI-K to the Golgi. As such, this study provides evidence for plant myosin recruitment and control of organelle movement.
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13
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Fernandes SA, Demetriades C. The Multifaceted Role of Nutrient Sensing and mTORC1 Signaling in Physiology and Aging. FRONTIERS IN AGING 2021; 2:707372. [PMID: 35822019 PMCID: PMC9261424 DOI: 10.3389/fragi.2021.707372] [Citation(s) in RCA: 49] [Impact Index Per Article: 12.3] [Reference Citation Analysis] [Abstract] [Key Words] [Grants] [Track Full Text] [Download PDF] [Figures] [Subscribe] [Scholar Register] [Received: 05/09/2021] [Accepted: 08/12/2021] [Indexed: 01/10/2023]
Abstract
The mechanistic Target of Rapamycin (mTOR) is a growth-related kinase that, in the context of the mTOR complex 1 (mTORC1), touches upon most fundamental cellular processes. Consequently, its activity is a critical determinant for cellular and organismal physiology, while its dysregulation is commonly linked to human aging and age-related disease. Presumably the most important stimulus that regulates mTORC1 activity is nutrient sufficiency, whereby amino acids play a predominant role. In fact, mTORC1 functions as a molecular sensor for amino acids, linking the cellular demand to the nutritional supply. Notably, dietary restriction (DR), a nutritional regimen that has been shown to extend lifespan and improve healthspan in a broad spectrum of organisms, works via limiting nutrient uptake and changes in mTORC1 activity. Furthermore, pharmacological inhibition of mTORC1, using rapamycin or its analogs (rapalogs), can mimic the pro-longevity effects of DR. Conversely, nutritional amino acid overload has been tightly linked to aging and diseases, such as cancer, type 2 diabetes and obesity. Similar effects can also be recapitulated by mutations in upstream mTORC1 regulators, thus establishing a tight connection between mTORC1 signaling and aging. Although the role of growth factor signaling upstream of mTORC1 in aging has been investigated extensively, the involvement of signaling components participating in the nutrient sensing branch is less well understood. In this review, we provide a comprehensive overview of the molecular and cellular mechanisms that signal nutrient availability to mTORC1, and summarize the role that nutrients, nutrient sensors, and other components of the nutrient sensing machinery play in cellular and organismal aging.
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Affiliation(s)
- Stephanie A. Fernandes
- Max Planck Institute for Biology of Ageing (MPI-AGE), Cologne, Germany
- Cologne Graduate School for Ageing Research (CGA), Cologne, Germany
| | - Constantinos Demetriades
- Max Planck Institute for Biology of Ageing (MPI-AGE), Cologne, Germany
- Cologne Graduate School for Ageing Research (CGA), Cologne, Germany
- University of Cologne, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), Cologne, Germany
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14
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Nüchel J, Tauber M, Nolte JL, Mörgelin M, Türk C, Eckes B, Demetriades C, Plomann M. An mTORC1-GRASP55 signaling axis controls unconventional secretion to reshape the extracellular proteome upon stress. Mol Cell 2021; 81:3275-3293.e12. [PMID: 34245671 PMCID: PMC8382303 DOI: 10.1016/j.molcel.2021.06.017] [Citation(s) in RCA: 39] [Impact Index Per Article: 9.8] [Reference Citation Analysis] [Abstract] [Key Words] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 10/20/2020] [Revised: 04/21/2021] [Accepted: 06/14/2021] [Indexed: 01/13/2023]
Abstract
Cells communicate with their environment via surface proteins and secreted factors. Unconventional protein secretion (UPS) is an evolutionarily conserved process, via which distinct cargo proteins are secreted upon stress. Most UPS types depend upon the Golgi-associated GRASP55 protein. However, its regulation and biological role remain poorly understood. Here, we show that the mechanistic target of rapamycin complex 1 (mTORC1) directly phosphorylates GRASP55 to maintain its Golgi localization, thus revealing a physiological role for mTORC1 at this organelle. Stimuli that inhibit mTORC1 cause GRASP55 dephosphorylation and relocalization to UPS compartments. Through multiple, unbiased, proteomic analyses, we identify numerous cargoes that follow this unconventional secretory route to reshape the cellular secretome and surfactome. Using MMP2 secretion as a proxy for UPS, we provide important insights on its regulation and physiological role. Collectively, our findings reveal the mTORC1-GRASP55 signaling hub as the integration point in stress signaling upstream of UPS and as a key coordinator of the cellular adaptation to stress.
mTORC1 phosphorylates GRASP55 directly at the Golgi in non-stressed cells mTORC1 inactivation by stress leads to GRASP55 dephosphorylation and relocalization GRASP55 relocalization to autophagosomes and MVBs drives UPS of selected cargo mTORC1-GRASP55 link cellular stress to changes in the extracellular proteome via UPS
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Affiliation(s)
- Julian Nüchel
- Max Planck Institute for Biology of Ageing (MPI-AGE), 50931 Cologne, Germany; University of Cologne, Faculty of Medicine and University Hospital Cologne, Center for Biochemistry, 50931 Cologne, Germany
| | - Marina Tauber
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Center for Biochemistry, 50931 Cologne, Germany
| | - Janica L Nolte
- University of Cologne, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), 50931 Cologne, Germany
| | | | - Clara Türk
- University of Cologne, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), 50931 Cologne, Germany
| | - Beate Eckes
- University of Cologne, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), 50931 Cologne, Germany; University of Cologne, Faculty of Medicine and University Hospital Cologne, Translational Matrix Biology, 50931 Cologne, Germany
| | - Constantinos Demetriades
- Max Planck Institute for Biology of Ageing (MPI-AGE), 50931 Cologne, Germany; University of Cologne, Cologne Excellence Cluster on Cellular Stress Responses in Aging-Associated Diseases (CECAD), 50931 Cologne, Germany.
| | - Markus Plomann
- University of Cologne, Faculty of Medicine and University Hospital Cologne, Center for Biochemistry, 50931 Cologne, Germany.
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15
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Sharma A, Chakraborty A, Jaganathan BG. Review of the potential of mesenchymal stem cells for the treatment of infectious diseases. World J Stem Cells 2021; 13:568-593. [PMID: 34249228 PMCID: PMC8246252 DOI: 10.4252/wjsc.v13.i6.568] [Citation(s) in RCA: 9] [Impact Index Per Article: 2.3] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Journal Information] [Submit a Manuscript] [Subscribe] [Scholar Register] [Received: 03/08/2021] [Revised: 04/07/2021] [Accepted: 06/03/2021] [Indexed: 02/06/2023] Open
Abstract
The therapeutic value of mesenchymal stem cells (MSCs) for the treatment of infectious diseases and the repair of disease-induced tissue damage has been explored extensively. MSCs inhibit inflammation, reduce pathogen load and tissue damage encountered during infectious diseases through the secretion of antimicrobial factors for pathogen clearance and they phagocytose certain bacteria themselves. MSCs dampen tissue damage during infection by downregulating the levels of pro-inflammatory cytokines, and inhibiting the excessive recruitment of neutrophils and proliferation of T cells at the site of injury. MSCs aid in the regeneration of damaged tissue by differentiating into the damaged cell types or by releasing paracrine factors that direct tissue regeneration, differentiation, and wound healing. In this review, we discuss in detail the various mechanisms by which MSCs help combat pathogens, tissue damage associated with infectious diseases, and challenges in utilizing MSCs for therapy.
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Affiliation(s)
- Amit Sharma
- Stem Cell and Cancer Biology Group, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, India
| | - Anuja Chakraborty
- Stem Cell and Cancer Biology Group, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, India
| | - Bithiah Grace Jaganathan
- Stem Cell and Cancer Biology Group, Department of Biosciences and Bioengineering, Indian Institute of Technology Guwahati, Guwahati 781039, India
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16
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Kim SH, Cho JH, Park BO, Park BC, Kim JH, Park SG, Kim S. Phosphorylation of REPS1 at Ser709 by RSK attenuates the recycling of transferrin receptor. BMB Rep 2021. [PMID: 33407999 PMCID: PMC8167248 DOI: 10.5483/bmbrep.2021.54.5.266] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Indexed: 11/20/2022] Open
Abstract
RalBP1 associated EPS domain containing 1 (REPS1) is conserved from Drosophila to humans and implicated in the endocytic system. However, an exact role of REPS1 remains largely unknown. Here, we demonstrated that mitogen activated protein kinase kinase (MEK)-p90 ribosomal S6 Kinase (RSK) signaling pathway directly phosphorylated REPS1 at Ser709 upon stimulation by epidermal growth factor (EGF) and amino acid. While REPS2 is known to be involved in the endocytosis of EGF receptor (EGFR), REPS1 knockout (KO) cells did not show any defect in the endocytosis of EGFR. However, in the REPS1 KO cells and the KO cells reconstituted with a non-phosphorylatable REPS1 (REPS1 S709A), the recycling of transferrin receptor (TfR) was attenuated compared to the cells reconstituted with wild type REPS1. Collectively, we suggested that the phosphorylation of REPS1 at S709 by RSK may have a role of the trafficking of TfR.
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Affiliation(s)
- Seong Heon Kim
- Department of Functional Genomics, KRIBB School of Biological Science, Korea University of Science and Technology, Daejeon 34113, Korea
- Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea
| | - Jin-hwa Cho
- Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea
| | - Bi-Oh Park
- Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea
- College of Pharmacy, Chungbuk National University, Cheongju 28160, Korea
| | - Byoung Chul Park
- Department of Functional Genomics, KRIBB School of Biological Science, Korea University of Science and Technology, Daejeon 34113, Korea
- Department of Proteome Structural Biology, KRIBB School of Biological Science, Korea University of Science and Technology, Daejeon 34113, Korea
| | - Jeong-Hoon Kim
- Department of Functional Genomics, KRIBB School of Biological Science, Korea University of Science and Technology, Daejeon 34113, Korea
- Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea
| | - Sung Goo Park
- Department of Functional Genomics, KRIBB School of Biological Science, Korea University of Science and Technology, Daejeon 34113, Korea
- Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea
| | - Sunhong Kim
- Disease Target Structure Research Center, Korea Research Institute of Bioscience and Biotechnology, Daejeon 34141, Korea
- Department of Biomolecular Science, KRIBB School of Biological Science, Korea University of Science and Technology, Daejeon 34113, Korea
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17
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Bhartiya A, Robinson I, Yusuf M, Botchway SW. Combining Multicolor FISH with Fluorescence Lifetime Imaging for Chromosomal Identification and Chromosomal Sub Structure Investigation. Front Mol Biosci 2021; 8:631774. [PMID: 33816553 PMCID: PMC8010142 DOI: 10.3389/fmolb.2021.631774] [Citation(s) in RCA: 1] [Impact Index Per Article: 0.3] [Reference Citation Analysis] [Abstract] [Key Words] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 11/20/2020] [Accepted: 02/02/2021] [Indexed: 11/25/2022] Open
Abstract
Understanding the structure of chromatin in chromosomes during normal and diseased state of cells is still one of the key challenges in structural biology. Using DAPI staining alone together with Fluorescence lifetime imaging (FLIM), the environment of chromatin in chromosomes can be explored. Fluorescence lifetime can be used to probe the environment of a fluorophore such as energy transfer, pH and viscosity. Multicolor FISH (M-FISH) is a technique that allows individual chromosome identification, classification as well as assessment of the entire genome. Here we describe a combined approach using DAPI as a DNA environment sensor together with FLIM and M-FISH to understand the nanometer structure of all 46 chromosomes in the nucleus covering the entire human genome at the single cell level. Upon DAPI binding to DNA minor groove followed by fluorescence lifetime measurement and imaging by multiphoton excitation, structural differences in the chromosomes can be studied and observed. This manuscript provides a blow by blow account of the protocol required to perform M-FISH-FLIM of whole chromosomes.
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Affiliation(s)
- Archana Bhartiya
- London Centre for Nanotechnology, University College London, London, United Kingdom.,Research Complex at Harwell Rutherford Appleton Laboratory, Didcot, United Kingdom
| | - Ian Robinson
- London Centre for Nanotechnology, University College London, London, United Kingdom.,Condensed Matter Physics and Materials Science Division, Brookhaven National Lab, Upton, NY, United States
| | - Mohammed Yusuf
- London Centre for Nanotechnology, University College London, London, United Kingdom.,Research Complex at Harwell Rutherford Appleton Laboratory, Didcot, United Kingdom.,Centre for Regenerative Medicine and Stem Cell Research, Aga Khan University, Karachi, Pakistan
| | - Stanley W Botchway
- Central Laser Facility, Science and Technology Facilities Council, Rutherford Appleton Laboratory, Oxon, United Kingdom
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18
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Ahmed AR, Candeo A, D'Abrantes S, Needham SR, Yadav RB, Botchway SW, Parker AW. Directly imaging the localisation and photosensitization properties of the pan-mTOR inhibitor, AZD2014, in living cancer cells. JOURNAL OF PHOTOCHEMISTRY AND PHOTOBIOLOGY. B, BIOLOGY 2020; 213:112055. [PMID: 33142217 PMCID: PMC7762844 DOI: 10.1016/j.jphotobiol.2020.112055] [Citation(s) in RCA: 4] [Impact Index Per Article: 0.8] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Figures] [Subscribe] [Scholar Register] [Received: 05/27/2020] [Revised: 09/24/2020] [Accepted: 10/12/2020] [Indexed: 12/17/2022]
Abstract
The range of cellular functions the mechanistic target of rapamycin (mTOR) protein performs makes it an attractive drug target for cancer therapy. However, the cellular localisation and mode of action of second generation inhibitors of mTOR is poorly understood despite the level of attention there is in targeting the mTOR protein. We have therefore studied the properties of the pan-mTOR inhibitor AZD2014, an ideal candidate to study because it is naturally fluorescent, characterising its photochemical properties in solution phase (DMSO, PBS and BSA) and within living cells, where it localises within both the nucleus and the cytoplasm but with different excited state lifetimes of 4.8 (+/- 0.5) and 3.9 (+/- 0.4) ns respectively. We measure the uptake of the inhibitor AZD2014 (7 μM) in monolayer HEK293 cells occurring with a half-life of 1 min but observe complex behaviour for 3D spheroids with the core of the spheroid showing a slower uptake and a slow biphasic behaviour at longer times. From a cellular perspective using fluorescence lifetime imaging microscopy AZD2014 was found to interact directly with GFP-tagged mTORC1 proteins including the downstream target, S6K1. We observe light sensitive behaviour of the cells containing AZD2014 which leads to cell death, in both monolayer and spheroids cells, demonstrating the potential of AZD2014 to act as a possible photodynamic drug under both single photon and multiphoton excitation and discuss its use as a photosensitizer. We also briefly characterise another pan-mTOR inhibitor, INK128.
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Affiliation(s)
- Abdullah R Ahmed
- Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK; Larch House, Woodlands Business Park, Breckland, Linford Wood, Milton Keynes MK14 6FG, UK
| | - Alessia Candeo
- Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK
| | - Sofia D'Abrantes
- Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK; CRUK/MRC Oxford Institute for Radiation Oncology, University of Oxford, Gray Laboratories, ORCRB Roosevelt Drive, Oxford OX3 7DQ, UK
| | - Sarah R Needham
- Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK
| | - Rahul B Yadav
- Evotec (UK) Ltd, 114 Innovation Drive, Milton Park, Abingdon, Oxfordshire OX14 4RZ, UK
| | - Stanley W Botchway
- Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK.
| | - Anthony W Parker
- Central Laser Facility, Science & Technology Facilities Council, Rutherford Appleton Laboratory, Harwell Campus, Didcot, Oxfordshire OX11 0QX, UK.
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19
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Martin-Fernandez ML. A brief history of the octopus imaging facility to celebrate its 10th anniversary. J Microsc 2020; 281:3-15. [PMID: 33111321 DOI: 10.1111/jmi.12974] [Citation(s) in RCA: 0] [Impact Index Per Article: 0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 08/06/2020] [Revised: 10/19/2020] [Accepted: 10/23/2020] [Indexed: 11/27/2022]
Abstract
Octopus (Optics Clustered to OutPut Unique Solutions) celebrated in June 2020 its 10th birthday. Based at Harwell, near Oxford, Octopus is an open access, peer reviewed, national imaging facility that offers successful U.K. applicants supported access to single molecule imaging, confocal microscopy, several flavours of superresolution imaging, light sheet microscopy, optical trapping and cryoscanning electron microscopy. Managed by a multidisciplinary team, Octopus has so far assisted >100 groups of U.K. and international researchers. Cross-fertilisation across fields proved to be a strong propeller of success underpinned by combining access to top-end instrumentation with a strong programme of imaging hardware and software developments. How Octopus was born, and highlights of the multidisciplinary output produced during its 10-year journey are reviewed below, with the aim of celebrating a myriad of collaborations with the U.K. scientific community, and reflecting on their scientific and societal impact.
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Affiliation(s)
- M L Martin-Fernandez
- Central Laser Facility, Research Complex at Harwell, Rutherford Appleton Laboratory, Harwell, Didcot, Oxford, U.K
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20
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Chen Y, Yang W, Shi X, Zhang C, Song G, Huang D. The Factors and Pathways Regulating the Activation of Mammalian Primordial Follicles in vivo. Front Cell Dev Biol 2020; 8:575706. [PMID: 33102482 PMCID: PMC7554314 DOI: 10.3389/fcell.2020.575706] [Citation(s) in RCA: 20] [Impact Index Per Article: 4.0] [Reference Citation Analysis] [Abstract] [Key Words] [Track Full Text] [Download PDF] [Figures] [Journal Information] [Subscribe] [Scholar Register] [Received: 06/24/2020] [Accepted: 09/07/2020] [Indexed: 11/13/2022] Open
Abstract
Mammalian ovaries consist of follicles as basic functional units. Each follicle comprised an innermost oocyte and several surrounding flattened granulosa cells. Unlike males, according to the initial size of the primordial follicle pool and the rate of its activation and depletion, a female's reproductive life has been determined early in life. Primordial follicles, once activated, will get into an irreversible process of development. Most follicles undergo atretic degeneration, and only a few of them could mature and ovulate. Although there are a lot of researches contributing to exploring the activation of primordial follicles, little is known about its underlying mechanisms. Thus, in this review, we collected the latest papers and summarized the signaling pathways as well as some factors involved in the activation of primordial follicles, hoping to lead to a more profound understanding of the cellular and molecular mechanisms of primordial follicle activation.
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Affiliation(s)
- Yao Chen
- Institute of Reproduction Health Research (Institute of Family Planning Research), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Weina Yang
- Institute of Reproduction Health Research (Institute of Family Planning Research), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Xu Shi
- Institute of Reproduction Health Research (Institute of Family Planning Research), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Chenlu Zhang
- Institute of Reproduction Health Research (Institute of Family Planning Research), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
| | - Ge Song
- NHC Key Laboratory of Male Reproduction and Genetics, Family Planning Research Institute of Guangdong Province, Guangzhou, China
| | - Donghui Huang
- Institute of Reproduction Health Research (Institute of Family Planning Research), Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China
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21
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Liu GY, Sabatini DM. mTOR at the nexus of nutrition, growth, ageing and disease. Nat Rev Mol Cell Biol 2020; 21:183-203. [PMID: 31937935 PMCID: PMC7102936 DOI: 10.1038/s41580-019-0199-y] [Citation(s) in RCA: 1661] [Impact Index Per Article: 332.2] [Reference Citation Analysis] [Abstract] [MESH Headings] [Grants] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Accepted: 11/26/2019] [Indexed: 12/21/2022]
Abstract
The mTOR pathway integrates a diverse set of environmental cues, such as growth factor signals and nutritional status, to direct eukaryotic cell growth. Over the past two and a half decades, mapping of the mTOR signalling landscape has revealed that mTOR controls biomass accumulation and metabolism by modulating key cellular processes, including protein synthesis and autophagy. Given the pathway's central role in maintaining cellular and physiological homeostasis, dysregulation of mTOR signalling has been implicated in metabolic disorders, neurodegeneration, cancer and ageing. In this Review, we highlight recent advances in our understanding of the complex regulation of the mTOR pathway and discuss its function in the context of physiology, human disease and pharmacological intervention.
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Affiliation(s)
- Grace Y Liu
- Whitehead Institute for Biomedical Research, Cambridge, MA, USA
- Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA
- Broad Institute, Cambridge, MA, USA
- The David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA, USA
| | - David M Sabatini
- Whitehead Institute for Biomedical Research, Cambridge, MA, USA.
- Department of Biology, Howard Hughes Medical Institute, Massachusetts Institute of Technology, Cambridge, MA, USA.
- Broad Institute, Cambridge, MA, USA.
- The David H. Koch Institute for Integrative Cancer Research at MIT, Cambridge, MA, USA.
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22
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Carroll B. Spatial regulation of mTORC1 signalling: Beyond the Rag GTPases. Semin Cell Dev Biol 2020; 107:103-111. [PMID: 32122730 DOI: 10.1016/j.semcdb.2020.02.007] [Citation(s) in RCA: 21] [Impact Index Per Article: 4.2] [Reference Citation Analysis] [Abstract] [Key Words] [MESH Headings] [Track Full Text] [Journal Information] [Subscribe] [Scholar Register] [Received: 01/08/2020] [Revised: 02/17/2020] [Accepted: 02/20/2020] [Indexed: 12/15/2022]
Abstract
The mechanistic (or mammalian) Target of Rapamycin Complex 1 (mTORC1) is a central regulator of cell growth and metabolism. By integrating mitogenic signals, mTORC1-dependent phosphorylation of substrates dictates the balance between anabolic, pro-growth and catabolic, recycling processes in the cell. The discovery that amino acids activate mTORC1 by promoting its translocation to the lysosome was a fundamental advance in the understanding of mTORC1 signalling. It has since become clear that the lysosome-cytoplasm shuttling of mTORC1 represents just one layer of spatial control of this signalling pathway. This review will focus on exploring the subcellular localisation of mTORC1 and its regulators to multiple sites within the cell. We will discuss how these spatially distinct regions such as endoplasmic reticulum, plasma membrane and the endosomal pathway co-operate to transduce nutrient availability to mTORC1, allowing for tight control of cell growth.
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Affiliation(s)
- Bernadette Carroll
- School of Biochemistry, Biomedical Sciences Building, University Walk, Bristol, BS8, United Kingdom.
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