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Development of a quantitative Correlative Light Electron Microscopy technique to study GLUT4 trafficking.

Hodgson L, Tavaré J, Verkade P - Protoplasma (2014)

Bottom Line: There are many different CLEM techniques, each having its own special advantages but also its technical challenges.It is however the biological question that (should) drive(s) the development and application of a specific CLEM technique in order to provide the answer.Here we describe the development of a CLEM technique that is based on the Tokuyasu cryo immuno-gold labelling technique that has allowed us to quantitatively study GLUT4 trafficking.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol, BS8 1TD, UK.

ABSTRACT
Correlative Light Electron Microscopy (CLEM) combines advantages of light microscopy and electron microscopy in one experiment to deliver information above and beyond the capability of either modality alone. There are many different CLEM techniques, each having its own special advantages but also its technical challenges. It is however the biological question that (should) drive(s) the development and application of a specific CLEM technique in order to provide the answer. Here we describe the development of a CLEM technique that is based on the Tokuyasu cryo immuno-gold labelling technique that has allowed us to quantitatively study GLUT4 trafficking.

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Insulin promotes the bulk recruitment of pXLG3.HA.GLUT4.GFP to the plasma membrane from vesicle clusters and tubular vesicular elements within the juxtanuclear region of the cell. Quantitative analysis of 3 T3-L1 adipocytes expressing pXLG3.HA.GLUT4.GFP in the presence and absence of insulin taken from two independent experiments. Gold labelling is expressed as % of total gold/membrane length. (Basal: N = 15 cells, n = 498 gold particles; insulin: N = 15, n = 592.) Graph represents mean ± SEM
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Fig7: Insulin promotes the bulk recruitment of pXLG3.HA.GLUT4.GFP to the plasma membrane from vesicle clusters and tubular vesicular elements within the juxtanuclear region of the cell. Quantitative analysis of 3 T3-L1 adipocytes expressing pXLG3.HA.GLUT4.GFP in the presence and absence of insulin taken from two independent experiments. Gold labelling is expressed as % of total gold/membrane length. (Basal: N = 15 cells, n = 498 gold particles; insulin: N = 15, n = 592.) Graph represents mean ± SEM

Mentions: Quantification of GLUT4 localisation in insulin and basal conditions supports these initial observations (Fig. 7). A 7-fold enrichment of cell surface GLUT4 and a 3-fold increase in GLUT4 in vesicles and tubules near the plasma membrane was observed in the insulin-stimulated state. This was accompanied by a 3-fold decrease in labelling of clusters and a 3-fold decrease in the amount of GLUT4 within the endosomes. Taken together, this data supports the evidence suggesting that GLUT4 translocates to the plasma membrane from a specialised storage pool consisting of a collection of small vesicles, roughly 70 nm in diameter.Fig. 7


Development of a quantitative Correlative Light Electron Microscopy technique to study GLUT4 trafficking.

Hodgson L, Tavaré J, Verkade P - Protoplasma (2014)

Insulin promotes the bulk recruitment of pXLG3.HA.GLUT4.GFP to the plasma membrane from vesicle clusters and tubular vesicular elements within the juxtanuclear region of the cell. Quantitative analysis of 3 T3-L1 adipocytes expressing pXLG3.HA.GLUT4.GFP in the presence and absence of insulin taken from two independent experiments. Gold labelling is expressed as % of total gold/membrane length. (Basal: N = 15 cells, n = 498 gold particles; insulin: N = 15, n = 592.) Graph represents mean ± SEM
© Copyright Policy - OpenAccess
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC3927059&req=5

Fig7: Insulin promotes the bulk recruitment of pXLG3.HA.GLUT4.GFP to the plasma membrane from vesicle clusters and tubular vesicular elements within the juxtanuclear region of the cell. Quantitative analysis of 3 T3-L1 adipocytes expressing pXLG3.HA.GLUT4.GFP in the presence and absence of insulin taken from two independent experiments. Gold labelling is expressed as % of total gold/membrane length. (Basal: N = 15 cells, n = 498 gold particles; insulin: N = 15, n = 592.) Graph represents mean ± SEM
Mentions: Quantification of GLUT4 localisation in insulin and basal conditions supports these initial observations (Fig. 7). A 7-fold enrichment of cell surface GLUT4 and a 3-fold increase in GLUT4 in vesicles and tubules near the plasma membrane was observed in the insulin-stimulated state. This was accompanied by a 3-fold decrease in labelling of clusters and a 3-fold decrease in the amount of GLUT4 within the endosomes. Taken together, this data supports the evidence suggesting that GLUT4 translocates to the plasma membrane from a specialised storage pool consisting of a collection of small vesicles, roughly 70 nm in diameter.Fig. 7

Bottom Line: There are many different CLEM techniques, each having its own special advantages but also its technical challenges.It is however the biological question that (should) drive(s) the development and application of a specific CLEM technique in order to provide the answer.Here we describe the development of a CLEM technique that is based on the Tokuyasu cryo immuno-gold labelling technique that has allowed us to quantitatively study GLUT4 trafficking.

View Article: PubMed Central - PubMed

Affiliation: School of Biochemistry, Medical Sciences Building, University of Bristol, University Walk, Bristol, BS8 1TD, UK.

ABSTRACT
Correlative Light Electron Microscopy (CLEM) combines advantages of light microscopy and electron microscopy in one experiment to deliver information above and beyond the capability of either modality alone. There are many different CLEM techniques, each having its own special advantages but also its technical challenges. It is however the biological question that (should) drive(s) the development and application of a specific CLEM technique in order to provide the answer. Here we describe the development of a CLEM technique that is based on the Tokuyasu cryo immuno-gold labelling technique that has allowed us to quantitatively study GLUT4 trafficking.

Show MeSH
Related in: MedlinePlus