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Article ; Online: Measuring EGFR plasma membrane density, stability, internalization, and recycling in alive adherent cells by cell surface ELISA.

Kazan, Jalal M / Pollato-Blanco, Ariadna / Lukacs, Gergely L / Pause, Arnim

STAR protocols

2022 Volume 3, Issue 3, Page(s) 101475

Abstract: EGFR cell surface density, stability, internalization, and recycling can be measured by cell surface ELISA (cs-ELISA). Performing this experiment on ice impedes receptor internalization; thus the physiological cell surface receptor levels can be measured ...

Abstract	EGFR cell surface density, stability, internalization, and recycling can be measured by cell surface ELISA (cs-ELISA). Performing this experiment on ice impedes receptor internalization; thus the physiological cell surface receptor levels can be measured by cs-ELISA. Cell surface EGFR levels are detected by measuring Amplex Red fluorescence intensity. Although cell surface receptor levels can be measured by flow cytometry, cs-ELISA does not include cell dissociation steps that might affect cell surface receptor levels. For complete details on the use and execution of this protocol, please refer to Kazan et al. (2019).
MeSH term(s)	Cell Membrane/metabolism ; Enzyme-Linked Immunosorbent Assay ; ErbB Receptors/metabolism ; Flow Cytometry ; Receptors, Cell Surface/metabolism
Chemical Substances	Receptors, Cell Surface ; ErbB Receptors (EC 2.7.10.1)
Language	English
Publishing date	2022-06-15
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't
ISSN	2666-1667
ISSN (online)	2666-1667
DOI	10.1016/j.xpro.2022.101475
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Folliculin: A Regulator of Transcription Through AMPK and mTOR Signaling Pathways.

Ramirez Reyes, Josué M J / Cuesta, Rafael / Pause, Arnim

Frontiers in cell and developmental biology

2021 Volume 9, Page(s) 667311

Abstract: Folliculin (FLCN) ...

Abstract	Folliculin (FLCN)
Language	English
Publishing date	2021-04-26
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2737824-X
ISSN	2296-634X
ISSN	2296-634X
DOI	10.3389/fcell.2021.667311
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Article ; Online: Biochemical Measurement of Glycogen: Method to Investigate the AMPK-Glycogen Relationship.

Possik, Elite / Pause, Arnim

Methods in molecular biology (Clifton, N.J.)

2018 Volume 1732, Page(s) 57–67

Abstract: Glycogen is a main carbohydrate energy storage primarily found in fungi and animals. It is a glucose polymer that comprises α(1-4) glycosidic linkages attaching UDP-glucose molecules linearly and α(1-6) linkages branching glucose chains every 8-10 ... ...

Abstract	Glycogen is a main carbohydrate energy storage primarily found in fungi and animals. It is a glucose polymer that comprises α(1-4) glycosidic linkages attaching UDP-glucose molecules linearly and α(1-6) linkages branching glucose chains every 8-10 molecules to the main backbone chain. Glycogen synthase, branching enzyme, and glycogen phosphorylase are key enzymes involved in glycogen synthesis and degradation. These enzymes are tightly regulated by upstream kinases and phosphatases that respond to hormonal cues in order to coordinate storage and degradation and meet the cellular and organismal metabolic needs. The 5'AMP-activated protein kinase (AMPK) is one of the main regulators of glycogen metabolism. Despite extensive research, the role of AMPK in glycogen synthesis and degradation remains controversial. Specifically, the level and duration of AMPK activity highly influence the outcome on glycogen reserves. Here, we describe a rapid and robust protocol to efficiently measure the levels of glycogen in vitro. We use the commercially available glycogen determination kit to hydrolyze glycogen into glucose, which is oxidized to form D-gluconic acid and hydrogen peroxide that react with the OxiRed/Amplex Red probe generating a product that could be detected either in a colorimetric or fluorimetric plate format. This method is quantitative and could be used to address the role of AMPK in glycogen metabolism in cells and tissues. Summary This chapter provides a quick and reliable biochemical quantitative method to measure glycogen in cells and tissues. Briefly, this method is based on the degradation of glycogen to glucose, which is then specifically oxidized to generate a product that reacts with the OxiRed probe with maximum absorbance at 570 nm. This method is very accurate and highly sensitive. In the notes of this chapter, we shed the light on important actions that should be followed to get reliable results. We also state advantages and disadvantages of this method in comparison to other glycogen measurement techniques.
MeSH term(s)	AMP-Activated Protein Kinases/metabolism ; Animals ; Cell Line, Tumor ; Colorimetry/instrumentation ; Colorimetry/methods ; Fluorometry/instrumentation ; Fluorometry/methods ; Glucose/chemistry ; Glucose/metabolism ; Glycogen/analysis ; Glycogen/metabolism ; Humans ; Hydrolysis ; Liver/metabolism ; Mice ; Muscle, Skeletal/metabolism ; Oxazines/chemistry ; Oxidation-Reduction ; Phosphorylation ; Reproducibility of Results ; Sensitivity and Specificity
Chemical Substances	Oxazines ; Amplex Red (119171-73-2) ; Glycogen (9005-79-2) ; AMP-Activated Protein Kinases (EC 2.7.11.31) ; Glucose (IY9XDZ35W2)
Language	English
Publishing date	2018-02-22
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Validation Studies
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-7598-3_4
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article: Glycogen: A must have storage to survive stressful emergencies.

Possik, Elite / Pause, Arnim

Worm

2016 Volume 5, Issue 2, Page(s) e1156831

Abstract: Mechanisms of adaptation to acute changes in osmolarity are fundamental for life. When exposed to hyperosmotic stress, cells and organisms utilize conserved strategies to prevent water loss and maintain cellular integrity and viability. The production of ...

Abstract	Mechanisms of adaptation to acute changes in osmolarity are fundamental for life. When exposed to hyperosmotic stress, cells and organisms utilize conserved strategies to prevent water loss and maintain cellular integrity and viability. The production of glycerol is a common strategy utilized by the nematode Caenorhabditis elegans (C. elegans) and many other organisms to survive hyperosmotic stress. Specifically, the transcriptional upregulation of glycerol-3-phosphate dehydrogenase, a rate-limiting enzyme in the production of glycerol, has been previously implicated in many model organisms. However, what fuels this massive and rapid production of glycerol upon hyperosmotic stress has not been clearly elucidated. We have recently discovered an AMPK-dependent pathway that mediates hyperosmotic stress resistance in C. elegans. Specifically, we demonstrated that the chronic activation of AMPK leads to glycogen accumulation, which under hyperosmotic stress exposure, is rapidly degraded to mediate glycerol production. Importantly, we demonstrate that this strategy is utilized by flcn-1 mutant C. elegans nematodes in an AMPK-dependent manner. FLCN-1 is the worm homolog of the human renal tumor suppressor Folliculin (FLCN) responsible for the Birt-Hogg-Dubé neoplastic syndrome. Here, we comment on the dual role for glycogen in stress resistance: it serves as an energy store and a fuel for osmolyte production. We further discuss the potential utilization of this mechanism by organisms in general and by human cancer cells in order to survive harsh environmental conditions and notably hyperosmotic stress.
Language	English
Publishing date	2016-03-04
Publishing country	United States
Document type	Comment ; Journal Article
ZDB-ID	2682460-7
ISSN	2162-4054 ; 2162-4046
ISSN (online)	2162-4054
ISSN	2162-4046
DOI	10.1080/21624054.2016.1156831
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Article: mTOR Pathways in Cancer and Autophagy.

Paquette, Mathieu / El-Houjeiri, Leeanna / Pause, Arnim

Cancers

2018 Volume 10, Issue 1

Abstract: TOR (target of rapamycin), an evolutionarily-conserved serine/threonine kinase, acts as a central regulator of cell growth, proliferation and survival in response to nutritional status, growth factor, and stress signals. It plays a crucial role in ... ...

Abstract	TOR (target of rapamycin), an evolutionarily-conserved serine/threonine kinase, acts as a central regulator of cell growth, proliferation and survival in response to nutritional status, growth factor, and stress signals. It plays a crucial role in coordinating the balance between cell growth and cell death, depending on cellular conditions and needs. As such, TOR has been identified as a key modulator of autophagy for more than a decade, and several deregulations of this pathway have been implicated in a variety of pathological disorders, including cancer. At the molecular level, autophagy regulates several survival or death signaling pathways that may decide the fate of cancer cells; however, the relationship between autophagy pathways and cancer are still nascent. In this review, we discuss the recent cellular signaling pathways regulated by TOR, their interconnections to autophagy, and the clinical implications of TOR inhibitors in cancer.
Language	English
Publishing date	2018-01-12
Publishing country	Switzerland
Document type	Journal Article ; Review
ZDB-ID	2527080-1
ISSN	2072-6694
ISSN	2072-6694
DOI	10.3390/cancers10010018
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Article ; Online: Structure and functions of His domain protein tyrosine phosphatase in receptor trafficking and cancer

Desrochers, Guillaume / Kazan, Jalal M / Pause, Arnim

Biochemistry and cell biology = Biochimie et biologie cellulaire

2018 Volume 97, Issue 1, Page(s) 68–72

Abstract: Cell surface receptors trigger the activation of signaling pathways to regulate key cellular processes, including cell survival and proliferation. Internalization, sorting, and trafficking of activated receptors, therefore, play a major role in the ... ...

Abstract	Cell surface receptors trigger the activation of signaling pathways to regulate key cellular processes, including cell survival and proliferation. Internalization, sorting, and trafficking of activated receptors, therefore, play a major role in the regulation and attenuation of cell signaling. Efficient sorting of endocytosed receptors is performed by the ESCRT machinery, which targets receptors for degradation by the sequential establishment of protein complexes. These events are tightly regulated and malfunction of ESCRT components can lead to abnormal trafficking and sustained signaling and promote tumor formation or progression. In this review, we analyze the modular domain organization of the alternative ESCRT protein HD-PTP and its role in receptor trafficking and tumorigenesis.
MeSH term(s)	Animals ; Endocytosis/physiology ; Endosomal Sorting Complexes Required for Transport/metabolism ; Humans ; Neoplasms/physiopathology ; Protein Transport ; Protein Tyrosine Phosphatases, Non-Receptor/chemistry ; Protein Tyrosine Phosphatases, Non-Receptor/metabolism ; Receptors, Cell Surface/metabolism ; Structure-Activity Relationship
Chemical Substances	Endosomal Sorting Complexes Required for Transport ; Receptors, Cell Surface ; PTPN23 protein, human (EC 3.1.3.48) ; Protein Tyrosine Phosphatases, Non-Receptor (EC 3.1.3.48)
Language	English
Publishing date	2018-06-07
Publishing country	Canada
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Review
ZDB-ID	54104-7
ISSN	1208-6002 ; 0829-8211
ISSN (online)	1208-6002
ISSN	0829-8211
DOI	10.1139/bcb-2017-0322
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Article ; Online: Measuring oxidative stress resistance of Caenorhabditis elegans in 96-well microtiter plates.

Possik, Elite / Pause, Arnim

Journal of visualized experiments : JoVE

2015 , Issue 99, Page(s) e52746

Abstract: Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and ... ...

Abstract	Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved. Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.
MeSH term(s)	Animals ; Caenorhabditis elegans/drug effects ; Caenorhabditis elegans/metabolism ; Caenorhabditis elegans Proteins/metabolism ; Hydrogen Peroxide/pharmacology ; Models, Animal ; Oxidative Stress/drug effects ; Oxidative Stress/physiology ; Paraquat/pharmacology ; Reactive Oxygen Species/metabolism ; Signal Transduction
Chemical Substances	Caenorhabditis elegans Proteins ; Reactive Oxygen Species ; Hydrogen Peroxide (BBX060AN9V) ; Paraquat (PLG39H7695)
Language	English
Publishing date	2015-05-09
Publishing country	United States
Document type	Journal Article ; Research Support, Non-U.S. Gov't ; Video-Audio Media
ISSN	1940-087X
ISSN (online)	1940-087X
DOI	10.3791/52746
Database	MEDical Literature Analysis and Retrieval System OnLINE

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Article ; Online: Single Cell Fluorescence Ratio Image Analysis for Studying ESCRT Function in Receptor Trafficking.

Kazan, Jalal M / Lukacs, Gergely L / Apaja, Pirjo M / Pause, Arnim

Methods in molecular biology (Clifton, N.J.)

2019 Volume 1998, Page(s) 93–103

Abstract: The endosomal sorting complexes required for transport (ESCRT) comprise a major trafficking pathway for plasma membrane proteins and are fundamental for ubiquitin-dependent cargo endocytosis. Here, we describe a method for studying the effect of ESCRT ... ...

Abstract	The endosomal sorting complexes required for transport (ESCRT) comprise a major trafficking pathway for plasma membrane proteins and are fundamental for ubiquitin-dependent cargo endocytosis. Here, we describe a method for studying the effect of ESCRT complexes on endo-lysosomal membrane trafficking and their role in receptor integrin α5β1 downregulation. Single cell fluorescence ratio image analysis (FRIA), using appropriate fluorescence probes, enables the measurement of dynamics of integrin α5β1 containing vesicles and represents a live cell-based method for studying the role of ESCRTs.
MeSH term(s)	Endosomal Sorting Complexes Required for Transport/chemistry ; Endosomal Sorting Complexes Required for Transport/metabolism ; Fluorescent Dyes/chemistry ; HeLa Cells ; Humans ; Image Processing, Computer-Assisted/methods ; Integrin alpha5beta1/chemistry ; Integrin alpha5beta1/metabolism ; Intracellular Membranes/chemistry ; Intracellular Membranes/metabolism ; Intravital Microscopy/methods ; Lysosomes/chemistry ; Lysosomes/metabolism ; Molecular Imaging/methods ; Molecular Probes/chemistry ; Single-Cell Analysis/methods
Chemical Substances	Endosomal Sorting Complexes Required for Transport ; Fluorescent Dyes ; Integrin alpha5beta1 ; Molecular Probes
Language	English
Publishing date	2019-02-28
Publishing country	United States
Document type	Journal Article
ISSN	1940-6029
ISSN (online)	1940-6029
DOI	10.1007/978-1-4939-9492-2_7
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Article: The dead phosphatases society: a review of the emerging roles of pseudophosphatases

Reiterer, Veronika / Pawłowski, Krzysztof / Desrochers, Guillaume / Pause, Arnim / Sharpe, Hayley J / Farhan, Hesso

FEBS journal. 2020 Oct., v. 287, no. 19

2020

Abstract: Phosphatases are a diverse family of enzymes, comprising at least 10 distinct protein folds. Like most other enzyme families, many have sequence variations that predict an impairment or loss of catalytic activity classifying them as pseudophosphatases. ... ...

Abstract	Phosphatases are a diverse family of enzymes, comprising at least 10 distinct protein folds. Like most other enzyme families, many have sequence variations that predict an impairment or loss of catalytic activity classifying them as pseudophosphatases. Research on pseudoenzymes is an emerging area of interest, with new biological functions repurposed from catalytically active relatives. Here, we provide an overview of the pseudophosphatases identified to date in all major phosphatase families. We will highlight the degeneration of the various catalytic sequence motifs and discuss the challenges associated with the experimental determination of catalytic inactivity. We will also summarize the role of pseudophosphatases in various diseases and discuss the major challenges and future directions in this field.
Keywords	catalytic activity ; enzymes ; society
Language	English
Dates of publication	2020-10
Size	p. 4198-4220.
Publishing place	John Wiley & Sons, Ltd
Document type	Article
Note	NAL-AP-2-clean ; REVIEW
ZDB-ID	2173655-8
ISSN	1742-4658 ; 1742-464X
ISSN (online)	1742-4658
ISSN	1742-464X
DOI	10.1111/febs.15431
Database	NAL-Catalogue (AGRICOLA)

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Article: Measuring oxidative stress resistance of Caenorhabditis elegans in 96-well microtiter plates

Possik, Elite / Pause, Arnim

Journal of visualized experiments. 2015 May 09, , no. 99

2015

Abstract	Oxidative stress, which is the result of an imbalance between production and detoxification of reactive oxygen species, is a major contributor to chronic human disorders, including cardiovascular and neurodegenerative diseases, diabetes, aging, and cancer. Therefore, it is important to study oxidative stress not only in cell systems but also using whole organisms. C. elegans is an attractive model organism to study the genetics of oxidative stress signal transduction pathways, which are highly evolutionarily conserved. Here, we provide a protocol to measure oxidative stress resistance in C. elegans in liquid. Briefly, ROS-inducing reagents such as paraquat (PQ) and H2O2 are dissolved in M9 buffer, and solutions are aliquoted in the wells of a 96 well microtiter plate. Synchronized L4/young adult C. elegans animals are transferred to the wells (5-8 animals/well) and survival is measured every hour until most worms are dead. When performing an oxidative stress resistance assay using a low concentration of stressors in plates, aging might influence the behavior of animals upon oxidative stress, which could lead to an incorrect interpretation of the data. However, in the assay described herein, this problem is unlikely to occur since only L4/young adult animals are being used. Moreover, this protocol is inexpensive and results are obtained in one day, which renders this technique attractive for genetic screens. Overall, this will help to understand oxidative stress signal transduction pathways, which could be translated into better characterization of oxidative stress-associated human disorders.
Keywords	Caenorhabditis elegans ; adults ; animal behavior ; diabetes ; genetics ; humans ; hydrogen peroxide ; liquids ; models ; neoplasms ; neurodegenerative diseases ; oxidative stress ; paraquat ; signal transduction ; stress tolerance
Language	English
Dates of publication	2015-0509
Size	p. e52746.
Publishing place	Journal of Visualized Experiments
Document type	Article
ZDB-ID	2259946-0
ISSN	1940-087X
ISSN	1940-087X
DOI	10.3791/52746
Database	NAL-Catalogue (AGRICOLA)

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