Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes

Morris, Silke; Geoghegan, Niall; Sadler, Jessica B. A.; Koester, Anna M.; Black, Hannah L; Laub, Marco; Miller, Lucy; Heffernan, Linda F.; Simpson, Jeremy C.; Mastick, Cynthia Corley; Cooper, Jonathan M.; Gadegaard, Nikolaj; Bryant, Nia J.; Gould, Gwyn W.

doi:10.7717/peerj.8751

Cited by 18 publications

(20 citation statements)

References 58 publications

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“…Both cell lines grow fast and can be used without the need for differentiation, which was challenging and time-consuming in previous studies [29]. In a recent study, it was demonstrated that HeLa cells expressing a similar GLUT4 fusion protein (HA-GLUT4-GFP) show comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes [39], confirming their applicability for our approach. In addition, these cells exhibit good adhesion properties on various surfaces that have been implemented for microscopy, which is especially important for TIRF microscopy.…”

Section: Discussionsupporting

confidence: 55%

Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

Schwarzinger

Lanzerstorfer

Neuhauser

et al. 2020

IJMS

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Due to the global rise of type 2 diabetes mellitus (T2DM) in combination with insulin resistance, novel compounds to efficiently treat this pandemic disease are needed. Screening for compounds that induce the translocation of glucose transporter 4 (GLUT4) from the intracellular compartments to the plasma membrane in insulin-sensitive tissues is an innovative strategy. Here, we compared the applicability of three fluorescence microscopy-based assays optimized for the quantitation of GLUT4 translocation in simple cell systems. An objective-type scanning total internal reflection fluorescence (TIRF) microscopy approach was shown to have high sensitivity but only moderate throughput. Therefore, we implemented a prism-type TIR reader for the simultaneous analysis of large cell populations grown in adapted microtiter plates. This approach was found to be high throughput and have sufficient sensitivity for the characterization of insulin mimetic compounds in live cells. Finally, we applied confocal microscopy to giant plasma membrane vesicles (GPMVs) formed from GLUT4-expressing cells. While this assay has only limited throughput, it offers the advantage of being less sensitive to insulin mimetic compounds with high autofluorescence. In summary, the combined implementation of different fluorescence microscopy-based approaches enables the quantitation of GLUT4 translocation with high throughput and high content.

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Section: Discussionsupporting

confidence: 55%

Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

Schwarzinger

Lanzerstorfer

Neuhauser

et al. 2020

IJMS

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“…For example, the up-regulation of soluble NSF attachment protein receptors (SNARE) interactions in vesicular transport at exhaustion following ZT0 exercise indicates the increase in a class of membrane-associated proteins, which, besides their involvement in neurotransmitter release (Dunant and Israel, 2000;Kasai et al, 2012), regulate GLUT4-containing vesicle trafficking (Cheatham, 2000). In line, the vesicle-associated membrane protein 3 and 8 (VAMP3, 8), syntaxin 6 and 8 (STX6, 8), synaptobrevin homolog YKT6 (YKT6), and of the synaptosomal-associated protein 23 (SNAP23), which are all key mediators of GLUT4 translocation to the cell surface were elevated at this timepoint (Bryant and Gould, 2011;Morris et al, 2020;Zong et al, 2011).…”

Section: Proteome and Secretome Changes Associated With Daytime Vs Nmentioning

confidence: 82%

Transcriptomic, proteomic and phosphoproteomic underpinnings of daily exercise performance and Zeitgeber activity of endurance training

Maier

Delezie

Westermark

et al. 2020

Preprint

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Timed physical activity might potentiate the health benefits of training. The underlying signaling events triggered by exercise at different times of the day are, however, poorly understood. Here, we found that time-dependent variations in maximal treadmill exercise capacity of naive mice were associated with energy stores, mostly hepatic glycogen levels. Importantly, running at different times of the day resulted in a vastly different activation of signaling pathways, e.g., related to stress response, vesicular trafficking, repair, and regeneration. Second, voluntary wheel running at the opposite phase of the dark, feeding period surprisingly revealed minimal Zeitgeber (i.e., synchronizing) activity of training. This integrated study provides important insights into the circadian regulation of endurance performance and the control of the circadian clock by exercise. These results are of high importance to understand circadian aspects of training design in athletes and the application of chrono-exercise-based interventions in patients.

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“…3T3-L1 murine adipocytes were purchased from ATCC (via LGC Standards, U.S.A. RRID:CVCL_0123) and grown and differentiated as outlined [ 34 , 41 ]. Stable lines of 3T3-L1 fibroblasts expressing HA-GLUT4-GFP had previously been generated in the lab [ 42 ]. Cells were incubated in a 10% CO 2 humid atmosphere incubator at 37°C.…”

Section: Methodsmentioning

confidence: 99%

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes

et al. 2022

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Insulin stimulates glucose transport in muscle and adipocytes. This is achieved by regulated delivery of intracellular glucose transporter (GLUT4)-containing vesicles to the plasma membrane where they dock and fuse, resulting in increased cell surface GLUT4 levels. Recent work identified a potential further regulatory step, in which insulin increases the dispersal of GLUT4 in the plasma membrane away from the sites of vesicle fusion. EFR3 is a scaffold protein that facilitates localisation of phosphatidylinositol 4-kinase type IIIa to the cell surface. Here we show that knockdown of EFR3 or phosphatidylinositol 4-kinase type IIIa impairs insulin-stimulated glucose transport in adipocytes. Using direct stochastic reconstruction microscopy, we also show that EFR3 knockdown impairs insulin stimulated GLUT4 dispersal in the plasma membrane. We propose that EFR3 plays a previously unidentified role in controlling insulin-stimulated glucose transport by facilitating dispersal of GLUT4 within the plasma membrane.

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Characterisation of GLUT4 trafficking in HeLa cells: comparable kinetics and orthologous trafficking mechanisms to 3T3-L1 adipocytes

Cited by 18 publications

References 58 publications

Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

Fluorescence Microscopy-Based Quantitation of GLUT4 Translocation: High Throughput or High Content?

Transcriptomic, proteomic and phosphoproteomic underpinnings of daily exercise performance and Zeitgeber activity of endurance training

EFR3 and phosphatidylinositol 4-kinase IIIα regulate insulin-stimulated glucose transport and GLUT4 dispersal in 3T3-L1 adipocytes

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